RESUMO
INTRODUCTION: The role of explosions and patient transport vehicles as sources and vectors of Gram-negative, multidrug-resistant organisms (MDROs) that predominate infections following lengthy evacuations after disasters due to natural hazards and in current war-trauma patients is unknown. HYPOTHESIS/PROBLEM: Damaged or heavily-used vehicles could be sources of the MDROs subsequently linked to nosocomial infections. METHODS: From January through May 2008 in Iraq, inside surfaces of heavily-used, tactical vehicles (Experimental Group) were sampled with sterile, pre-moistened swabs. Swabs, along with positive and negative controls, were shipped to the reference laboratory in Washington, DC, where they underwent culture, identification and susceptibility testing, and pulsed-field gel electrophoresis. Multidrug-resistant organisms were defined according to the standard Centers for Disease Control and Prevention definitions. High risk organisms (HROs) were defined as susceptible E. coli, A. baumannii, P. aeruginosa, Enterobacter spp, or Klebsiella spp. Concurrently, new counterparts (Control Group) were similarly surveyed in a storage lot in Georgia, USA. Groups were compared using the Chi-squared test. RESULTS: One hundred thirty-nine consecutive vehicles including all available ambulances were sampled, yielding 153 swabs. Nineteen were lost or damaged during shipping. Seventy-nine swabs yielded growth of one or more Gram-negative bacteria. The amount and genotype of MDROs in heavily-used vehicles, including those involved in roadside bombings, were compared to control vehicles and to strains isolated from wounds and environmental surfaces at the base hospital. Predominant organisms included P. agglomerans (34%), S. flexneri (8%), E. vulneris (6%), Pseudomonas sp. (6%), and K. pneumonia (6%). No MDROs were isolated. Thirteen vehicles (eight of 94 experimental and five of 45 control) yielded HRO. There was no difference in contamination rates (P = .63). No HROs were isolated from ambulances. No clonal association existed between vehicle and hospital strains. CONCLUSION: Given the implications that this knowledge gap has on military and civilian prehospital reservoirs of infection, further study is warranted to confirm these findings and identify targets for preventive intervention throughout civilian disaster and military casualty evacuation chains.
Assuntos
Ambulâncias , Infecções Bacterianas/etiologia , Infecção Hospitalar/etiologia , Desastres , Reservatórios de Doenças , Farmacorresistência Bacteriana Múltipla , Contaminação de Equipamentos , Explosões , Infecções Bacterianas/prevenção & controle , Estudos de Casos e Controles , Infecção Hospitalar/prevenção & controle , Humanos , Guerra do Iraque 2003-2011 , Medicina Militar , Risco , Estados UnidosRESUMO
BACKGROUND: Effective prophylaxis and treatment for infections caused by biological threat agents (BTA) rely upon early diagnosis and rapid initiation of therapy. Most methods for identifying pathogens in body fluids and tissues require that the pathogen proliferate to detectable and dangerous levels, thereby delaying diagnosis and treatment, especially during the prelatent stages when symptoms for most BTA are indistinguishable flu-like signs. METHODS: To detect exposures to the various pathogens more rapidly, especially during these early stages, we evaluated a suite of host responses to biological threat agents using global gene expression profiling on complementary DNA arrays. RESULTS: We found that certain gene expression patterns were unique to each pathogen and that other gene changes occurred in response to multiple agents, perhaps relating to the eventual course of illness. Nonhuman primates were exposed to some pathogens and the in vitro and in vivo findings were compared. We found major gene expression changes at the earliest times tested post exposure to aerosolized B. anthracis spores and 30 min post exposure to a bacterial toxin. CONCLUSION: Host gene expression patterns have the potential to serve as diagnostic markers or predict the course of impending illness and may lead to new stage-appropriate therapeutic strategies to ameliorate the devastating effects of exposure to biothreat agents.
Assuntos
Bacillus anthracis/imunologia , Armas Biológicas , Perfilação da Expressão Gênica/métodos , Leucócitos Mononucleares/imunologia , Análise de Variância , Animais , Antraz/genética , Exposição Ambiental , Expressão Gênica , Humanos , Macaca mulatta , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fatores de TempoAssuntos
Bacteriófagos , Peste/terapia , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos BALB CAssuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/classificação , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter calcoaceticus/efeitos dos fármacos , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Farmacorresistência Bacteriana Múltipla , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Acinetobacter calcoaceticus/classificação , Acinetobacter calcoaceticus/genética , Acinetobacter calcoaceticus/isolamento & purificação , Análise por Conglomerados , DNA Bacteriano/genética , Genótipo , Humanos , Análise em Microsséries , Testes de Sensibilidade Microbiana , Medicina Naval , Fenótipo , Estados UnidosRESUMO
BACKGROUND: Infections by pan-drug resistant Acinetobacter baumannii plague military and civilian healthcare systems. Previous A. baumannii pan-genomic studies used modest sample sizes of low diversity and comparisons to a single reference genome, limiting our understanding of gene order and content. A consensus representation of multiple genomes will provide a better framework for comparison. A large-scale comparative study will identify genomic determinants associated with their diversity and adaptation as a successful pathogen. RESULTS: We determine draft-level genomic sequence of 50 diverse military isolates and conduct the largest bacterial pan-genome analysis of 249 genomes. The pan-genome of A. baumannii is open when the input genomes are normalized for diversity with 1867 core proteins and a paralog-collapsed pan-genome size of 11,694 proteins. We developed a novel graph-based algorithm and use it to assemble the first consensus pan-chromosome, identifying both the order and orientation of core genes and flexible genomic regions. Comparative genome analyses demonstrate the existence of novel resistance islands and isolates with increased numbers of resistance island insertions over time, from single insertions in the 1950s to triple insertions in 2011. Gene clusters responsible for carbon utilization, siderophore production, and pilus assembly demonstrate frequent gain or loss among isolates. CONCLUSIONS: The highly variable and dynamic nature of the A. baumannii genome may be the result of its success in rapidly adapting to both abiotic and biotic environments through the gain and loss of gene clusters controlling fitness. Importantly, some archaic adaptation mechanisms appear to have reemerged among recent isolates.
Assuntos
Acinetobacter baumannii/genética , Cromossomos Bacterianos , Genoma Bacteriano , Genômica/métodos , Acinetobacter baumannii/isolamento & purificação , Acinetobacter baumannii/patogenicidade , Algoritmos , Ordem dos Genes , Genes Essenciais , Ilhas Genômicas , Humanos , Redes e Vias Metabólicas/genética , Militares , Virulência/genéticaRESUMO
A multiplex TaqMan real-time PCR to detect carbapenem-hydrolysing class D ß-lactamases (bla(OXA-23)-like, bla(OXA-24/40)-like, bla(OXA-51)-like and bla(OXA-58)-like genes) was developed and evaluated for early detection of imipenem (IMP) resistance in clinically significant Acinetobacter baumannii isolates. Well-characterized strains of A. baumannii were used as positive controls and non-Acinetobacter strains were used to assess specificity. Analytical sensitivity was quantified by comparison with the number of bacterial c.f.u. Forty of 46 (87â%) clinically significant and IMP-resistant A. baumannii isolates were positive for the bla(OXA-23)-like gene, and one isolate (2â%) was positive for the bla(OXA-58)-like gene. The bla(OXA-24/40)-like gene was not detected in any of the 46 IMP-resistant strains and the bla(OXA-51)-like gene was identified in both IMP-resistant and non-resistant A. baumannii. All 11 non-Acinetobacter bacteria produced a negative result for each of the four bla(OXA) genes. This assay was able to detect as few as 10 c.f.u. per assay. This real-time PCR method demonstrated rapid detection of OXA-like carbapenem resistance in A. baumannii in comparison with phenotypic susceptibility testing methodology. This method could be adapted to a multiplexed single reaction for rapid detection of genes associated with carbapenem resistance in A. baumannii and potentially other clinically significant multidrug-resistant Gram-negative bacteria.
Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , beta-Lactamases/metabolismoRESUMO
BACKGROUND: Gram-negative multidrug-resistant (MDR) bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq. METHODOLOGY/PRINCIPAL FINDINGS: In this study, all MDR Enterobacteriaceae (n = 38) and randomly selected non-MDR counterparts (n = 41) isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥ 3) plasmids compared to their non-MDR counterparts, which carried ≤ 2 plasmids (p<0.01). Various large plasmids (~52 to 100 kb) from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA), ß-lactam (bla(TEM1), bla(AMPC), bla(CTX-M-15), bla(OXA-1), bla(VIM-2) and bla(SHV)), sulfamethoxazole/trimethoprim (sul/dfr), tetracycline (tet) and chloramphenicol (cat) resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N) were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates. CONCLUSIONS/SIGNIFICANCE: This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary relationship with other parts of world. The large plasmids, carrying resistance genes and transfer-associated genes, may be potential factors for regional dissemination of antibiotic resistance.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Hospitais/estatística & dados numéricos , Plasmídeos/genética , Vigilância da População , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Genes Bacterianos/genética , Humanos , Iraque/epidemiologia , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos , Estudos Prospectivos , Replicon/genética , Transformação GenéticaRESUMO
OBJECTIVE: To investigate potential sources of gram-negative multidrug-resistant organisms (MDROs) in a deployed US military healthcare facility. DESIGN: Active surveillance. METHODS: Swab sampling of patients, hospital personnel, and environmental surfaces was performed before the opening of a new medical treatment facility in Iraq and then serially for the next 6 months. Multidrug resistant isolates were genotypically characterized using pulsed-field gel electrophoresis (PFGE). Univariate and multivariate analysis were performed to evaluate associations between patient characteristics and MDRO carriage. SETTING: Deployed US military medical facility. RESULTS: A total of 1,348 samples were obtained, yielding 654 isolates, 42 of which were MDROs. One hundred fifty-eight patients were sampled; swabs from 18 patients yielded 29 MDR isolates. Host nation patients comprised 89% of patients with MDROs and 37% of patients without MDROs (P < .001). Host nation patient status was also significantly associated with MDRO carriage in multivariate logistic regression analysis (adjusted odds ratio, 2.9; confidence interval, 1.3-6.3; P = .009). Bacteria with PFGE patterns matching those recovered from host nation patients were later isolated from environmental surfaces including recovery room patient monitors and the trauma bay floor. CONCLUSIONS: At this facility, MDRO isolation was predominantly obtained from newly admitted host nation patients, which may reflect baseline colonization with MDROs in the community. Patient MDRO carriage was linked to subsequent environmental contamination. These findings support intensive infection control efforts in forward deployed facilities.
Assuntos
Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Hospitais Militares , Militares , Adolescente , Adulto , Axila/microbiologia , Portador Sadio , Criança , Pré-Escolar , Infecção Hospitalar/epidemiologia , Eletroforese em Gel de Campo Pulsado , Contaminação de Equipamentos , Feminino , Infecções por Bactérias Gram-Negativas/epidemiologia , Virilha/microbiologia , Humanos , Pacientes Internados , Iraque , Masculino , Pessoa de Meia-Idade , Recursos Humanos em Hospital , Estados Unidos , Adulto JovemRESUMO
BACKGROUND: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. CONCLUSIONS/SIGNIFICANCE: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.
Assuntos
Bacteriófagos/fisiologia , Mutação , Receptores Virais/metabolismo , Yersinia pestis/genética , Yersinia pestis/virologia , Animais , Bacteriófagos/metabolismo , Feminino , Lipopolissacarídeos/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Peste/terapia , Peste/virologia , Transporte Proteico , Receptores Virais/genética , Especificidade da Espécie , Yersinia pestis/metabolismo , Yersinia pestis/patogenicidadeRESUMO
OBJECTIVE: To determine the anatomic sites and natural history of colonization with gram-negative multidrug-resistant organisms (MDROs). DESIGN: Prospective, longitudinal cohort study. SETTING: Walter Reed Army Medical Center, a 236-bed tertiary care center in Washington, DC. PATIENTS: Deployed subjects (ie, inpatients medically evacuated from Iraq or Afghanistan) or nondeployed subjects admitted to the same hospital. METHODS: Consenting patients had 6 anatomic sites cultured every 3 days for 2 weeks and then weekly. Gram-negative organisms resistant to 3 or more classes of antibiotics were considered MDROs. Isolates were genotyped using pulsed-field gel electrophoresis. Clinical data, data on antibiotic use, and clinical culture results were collected. RESULTS: Of 60 deployed subjects, 14 (23%) were colonized with an MDRO at admission, and 13 (22%) had incident colonization during hospitalization. The groin was the most sensitive anatomic site for detecting MDRO colonization, and all but one subject remained colonized for the duration of their hospitalization. Sixty percent of subjects with incident Acinetobacter colonization and 25% of subjects with incident Klebsiella colonization had strains that were related to those isolated from other subjects. Of 60 nondeployed subjects, 5 (8%) were colonized with an MDRO at admission; all had recent healthcare contact, and 1 nondeployed subject had an isolate related to a strain recovered from a deployed subject. CONCLUSIONS: Colonization with gram-negative MDROs is common among patients with war-related trauma admitted to a military hospital and also occurs among nondeployed patients with recent healthcare contact. The groin is the most sensitive anatomic site for active surveillance, and spontaneous decolonization is rare.
Assuntos
Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/isolamento & purificação , Hospitalização/estatística & dados numéricos , Adulto , Antibacterianos/farmacologia , Estudos de Coortes , District of Columbia , Feminino , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Virilha/microbiologia , Hospitais Militares , Humanos , Masculino , Testes de Sensibilidade Microbiana , Especificidade de Órgãos , Prevalência , Estudos Prospectivos , Reto/microbiologia , Estados Unidos , Adulto JovemRESUMO
Yersinia pestis is the causative agent of plague, which diverged from Yersinia pseudotuberculosis within the past 20,000 years. Although these two species share a high degree of homology at the DNA level (>90%), they differ radically in their pathogenicity and transmission. In this review, we briefly outline the known virulence factors that differentiate these two species and emphasize genetic studies that have been conducted comparing Y. pestis and Y. pseudotuberculosis. These comparisons have led to a better understanding of the genetic contributions to the differences in the virulence and pathogenicity between these two organisms and have generated information that can be applied in future diagnostic and vaccine development. Comparison of the genetic differences between Y. pestis and Y. pseudotuberculosis has also lent insight into the emergence of acute pathogens from organisms causing milder diseases.
Assuntos
Peste/microbiologia , Yersinia pestis/genética , Yersinia pestis/patogenicidade , Animais , Genes Bacterianos , Genômica , Humanos , Modelos Biológicos , Mutagênese , Plasmídeos/genética , Pesquisa/tendências , Especificidade da Espécie , Virulência/genética , Yersinia pestis/metabolismo , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/patogenicidade , Infecções por Yersinia pseudotuberculosis/microbiologiaRESUMO
The pH 6 antigen (pH 6 Ag; PsaA) of Yersinia pestis has been shown to be a virulence factor. In this study, we set out to investigate the possible function of Y. pestis PsaA in a host cell line, RAW264.7 mouse macrophages, in order to better understand the role it might play in virulence. Y. pestis KIM5 derivatives with and without the pCD1 plasmid and their psaA isogenic counterparts and Escherichia coli HB101 and DEta5alpha carrying a psaA clone or a vector control were used for macrophage infections. Macrophage-related bacteria and gentamicin-resistant intracellular bacteria generated from plate counting and direct microscopic examinations were used to evaluate these RAW264.7 macrophage infections. Y. pestis psaA isogenic strains did not show any significant difference in their abilities to associate with or bind to mouse macrophage cells. However, expression of psaA appeared to significantly reduce phagocytosis of both Y. pestis and E. coli by mouse macrophages (P < 0.05). Furthermore, we found that complementation of psaA mutant Y. pestis strains could completely restore the ability of the bacteria to resist phagocytosis. Fluorescence microscopy following differential labeling of intracellular and extracellular Y. pestis revealed that significantly lower numbers of psaA-expressing bacteria were located inside the macrophages. Enhanced phagocytosis resistance was specific for bacteria expressing psaA and did not influence the ability of the macrophages to engulf other bacteria. Our data demonstrate that Y. pestis pH 6 Ag does not enhance adhesion to mouse macrophages but rather promotes resistance to phagocytosis.
Assuntos
Antígenos de Bactérias/fisiologia , Cápsulas Bacterianas/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/fisiologia , Fagocitose , Yersinia pestis/patogenicidade , Animais , Aderência Bacteriana , Linhagem Celular , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Microscopia de FluorescênciaRESUMO
Yersinia pestis, the causative agent of deadly plague, is considered a reemerging infectious disease and a significant biological terrorism threat. The present project focused on epidemiological investigation of the genetic variability of well-documented strains of Y. pestis from the United States by pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis with insertion sequences IS100 and IS285 as probes. We examined 37 U.S. Y. pestis strains and isolates of a single ribotype, ribotype B, recovered between 1939 and 1998 from patients, animals, and fleas. Our results showed that all isolates had similar PFGE patterns, but minor differences such as missing, additional, and shifted bands were found among almost all strains if they came from different parent strains. The 37 strains and isolates were divided into 26 PFGE types. RFLP analysis with IS100 as a probe divided these strains and isolates into 16 types, with 43% belonging to IS100 type 1. Typing with IS285 as a probe was less specific and led to only four RFLP types, with 81% belonging to type 1. Similarity analysis with BioNumerics software showed that all strains shared >or=80, 86, and 91% similarities on dendrograms prepared from digitized PFGE, IS100 RFLP analysis, and IS285 RFLP analysis images, respectively. Our results demonstrate that PFGE offers an increased ability to discriminate between strains (Simpson's index of diversity, 0.98) and therefore can significantly improve epidemiological studies related to the origin of new plague isolates.