First Report of an OXA-23 Carbapenemase-Producing Acinetobacter baumannii Clinical Isolate Related to Tn2006 in Spain

A carbapenem-resistant Acinetobacter baumannii clinical isolate belonging to European clone II and sequence type 2 was recovered from a patient in the Son Espases hospital in Mallorca, Spain. Genetic analysis showed the presence of the bla_OXA-23 gene in association with the widely disseminated transposon Tn2006. This is the first reported identification of A. baumannii carrying bla_OXA-23 in Spain.     

First Report of NDM-1-Producing Acinetobacter baumannii Sequence Type 25 in Brazil

New Delhi metallo-β-lactamase 1 (NDM-1) was first identified in Brazil in Enterobacter hormaechei and Providencia rettgeri in 2013. Here, we describe the first case of NDM-1-producing Acinetobacter baumannii sequence type 25 isolated from the urinary tract of a 71-year-old man who died of multiple complications, including A. baumannii infection. The NDM-1 gene was detected by quantitative PCR, and its sequence confirmed its presence in an ∼100-kb plasmid.     

First Description of Two Sequence Type 2 Acinetobacter baumannii Isolates Carrying OXA-23 Carbapenemase in Pagellus acarne Fished from the Mediterranean Sea near Bejaia,Algeria

To determine the occurrence of carbapenem-resistant Acinetobacter baumannii in fish fished from the Mediterranean Sea near the Bejaia coast (Algeria), we studied 300 gills and gut samples that had been randomly and prospectively collected during 1 year. After screening on selective agar media, using PCR arrays and whole-genome sequencing, we identified for the first time two OXA-23-producing A. baumannii strains belonging to the widespread sequence type 2 (ST2)/international clone II and harboring aminoglycoside-modifying enzymes [aac(6′)-Ib and aac(3′)-I genes].     

Clinical and Epidemiological Significance of Carbapenem Resistance in Acinetobacter baumannii Infections

Carbapenems are considered the treatment of choice for Acinetobacter baumannii infections. Many facilities implement preventive measures toward only carbapenem-resistant A. baumannii (CRAB). However, the independent role of the carbapenem resistance determinant on patient outcomes remains controversial. In a 6-year analysis of adults with A. baumannii bloodstream infection (BSI), the outcomes of 149 CRAB isolates were compared to those of 91 patients with carbapenem-susceptible A. baumannii. In bivariable analyses, CRAB BSIs were significantly associated with worse outcomes and with a delay in the initiation of appropriate antimicrobial therapy (DAAT). However, in multivariable analyses, carbapenem resistance status was no longer associated with poor outcomes, while DAAT remained an independent predictor. The epidemiological significance of A. baumannii should not be determined by its resistance to carbapenems.     

耐碳青霉烯的多重耐药鲍曼不动杆菌分子流行病学研究

目的调查沈阳地区多重耐药鲍曼不动杆菌（Multi—drug Resistant Acinetobacter Baumannii,MDR—Aba）的同源性,了解是否存在该类耐药株的克隆流行。方法收集2007年10月-2008年12月沈阳军区总医院住院患者临床分离出的MDR—Aba40株。用K—B纸片扩散法检测其对临床常用抗生素的敏感性,用脉冲场凝胶电泳（Pulsed—field gel electrophoresis,PFGE）对所选菌株进行分子分型。结果脉冲场凝胶电泳结果显示,40株MDR—Aba茵株分为A,B,C三个型,其中A型包括A1（28株主要分布于呼吸、神经外、急诊、心外等8个病区）,A2（2株,分布于呼吸、急诊病区）,A3（6株,分布于神经外、呼吸等病区）三个亚型;B型、C型各2株,主要分布于急诊病区。结论调查期间该院部分科室有A型MDR—Aba菌株克隆流行,主要为A1型,同时有少量的A2,A3亚型;偶见B型、C型。可见及早采取有力的院内感染防控措施,是防控耐碳青霉烯MDR—Aba暴发流行的当务之急。     

Protective Effect of a Synbiotic against Multidrug-Resistant Acinetobacter baumannii in a Murine Infection Model

This study investigated the ability of the probiotic Bifidobacterium breve strain Yakult (BbY) to protect against infection, as well as the potentiation of BbY activity by the synbiotic combination of BbY and prebiotic galactooligosaccharides (GOS). The study employed a mouse model of lethal intestinal multidrug-resistant Acinetobacter baumannii (MDRAb) infection. The endogenous intestinal microbiota was disrupted by the administration of multiple antibiotics, causing the loss of endogenous Bifidobacterium. Oral infection of these mice with MDRAb resulted in marked growth of this organism. Additional treatment of the infected mice with a sublethal dose of 5-fluorouracil (5-FU) induced systemic invasion by MDRAb and subsequent animal death. The continuous oral administration of BbY increased the survival rate and inhibited the intestinal growth and invasion by MDRAb in the infection model. Disruptions of the intestinal environment and barrier function in the infected mice were attenuated by BbY. Protection against the MDRAb infection was markedly potentiated by a synbiotic combination of BbY and GOS, although GOS by itself did not provide protection. Negative correlations were observed between intestinal MDRAb and BbY counts or acetic acid levels; positive correlations were observed between acetic acid levels and intestinal epithelium expression of tight-junction-related genes. These results demonstrated that the probiotic and synbiotic markedly potentiated protection against fatal intestinal infection caused by a multidrug-resistant bacterium. Probiotics and synbiotics are presumed to provide protection by compensation for the disrupted indigenous populations, thereby maintaining the intestinal environments and barrier functions otherwise targeted during opportunistic infection by MDRAb.     

Multiple Genetic Mutations Associated with Polymyxin Resistance in Acinetobacter baumannii

We studied polymyxin B resistance in 10 pairs of clinical Acinetobacter baumannii isolates, two of which had developed polymyxin B resistance in vivo. All polymyxin B-resistant isolates had lower growth rates than and substitution mutations in the lpx or pmrB gene compared to their parent isolates. There were significant differences in terms of antibiotic susceptibility and genetic determinants of resistance in A. baumannii isolates that had developed polymyxin B resistance in vivo compared to isolates that had developed polymyxin B resistance in vitro.     

儿童鲍曼不动杆菌产IMP-4及OXA-23型碳青霉烯酶分子流行病学

目的 研究武汉地区儿童分离CRAB中获得性碳青霉烯酶分子流行病学特征.方法 收集2008年12月至2009年5月期间武汉市儿童医院急救科、新生儿内科、心胸外科、骨外科、呼吸内科及肾内科住院患儿分离的非重复CRAB 40株,使用生物梅里埃公司生产的GNS-142检测菌株的MIC值,PFGE分析耐药菌株的同源性.采用PCR扩增KPC、IMP、GIM、SPM、SIM、OXA-23、VIM 7种获得性碳青霉烯酶基因及整合酶基因,测序确定基因型,质粒接合转移试验分析细菌耐药性的转移方式,Southern blot试验定位耐药基因.结果 40株CRAB对庆大霉素、妥布霉素、阿米卡星、环丙沙星、左氧氟沙星、复方磺胺甲(口恶)唑的敏感率分别为20%、5%、93%、93%、95%及23%;PFGE分型共检出11种克隆株,其中A型29株,B型2株,C～K型各1株.11株同时产IMP-4型MBL及OXA-23酶,26株只产OXA-23酶;未检出GIM、SPM、SIM、KPC和VIM型碳青霉烯酶基因.36株鲍曼不动杆菌携带Ⅰ类整合子(Int1).Southern blot表明Int1、IMP-4及OXA-23型定位于细菌染色体上.未见质粒接合转移试验阳性菌株.结论 A型克隆耐药株为本地区最常见CRAB菌株,OXA-23酶及IMP-4型是CRAB中主要的获得性碳青霉烯酶类型,其中以OXA-23型最为常见,Int1介导的OXA-23及IMP-4基因的水平传播及A型耐药克隆株的传播是本地区鲍曼不动杆菌中碳青霉烯类耐药性传播的主要方式.     

2型糖尿病患者合并泌尿系感染的病原菌分布及耐药性分析

[目的]了解本地区2型糖尿病（T2DM）患者合并泌尿系感染的病原菌分布以及耐药情况,为临床治疗提供可靠依据,指导临床合理用药.[方法]对2012年1月至2013年12月期间本院625例T2DM住院患者的尿细菌培养与药敏结果进行回顾性分析.[结果]在625例T2DM患者无菌中段尿中,捡出病原菌122株,革兰阴性杆菌占75.41％,其中无症状感染患者占43.58％.大肠埃希菌和肺炎克雷伯菌对氟喹诺酮类抗菌药物的耐药性大于60％.[结论]T2DM患者合并泌尿系感染,临床治疗应积极控制血糖,及早发现,根据药物敏感试验选用抗生素治疗.     

CraA,a Major Facilitator Superfamily Efflux Pump Associated with Chloramphenicol Resistance in Acinetobacter baumannii

Acinetobacter baumannii has been increasingly associated with hospital-acquired infections, and the presence of multidrug resistance strains is of great concern to clinicians. A. baumannii is thought to possess a great deal of intrinsic resistance to several antimicrobial agents, including chloramphenicol, although the mechanisms involved in such resistance are not well understood. In this work, we have identified a major facilitator superfamily efflux pump present in most A. baumannii strains, displaying strong substrate specificity toward chloramphenicol.Acinetobacter baumannii is an important opportunistic pathogen often associated with severe nosocomial infections, such as pneumonia, bacteremia, and secondary meningitis, especially in intensive care units. In the last few decades, A. baumannii infections have become a serious health problem due to the emergence of multidrug-resistant (MDR) phenotypes which, therefore, restrain antimicrobial therapy to just a few agents, mainly carbapenems (2, 22).Antimicrobial resistance can be due to several distinct mechanisms, being that MDR phenotypes are related to the acquisition of genetic elements carrying different resistant determinants or to decreased membrane permeability, together with expression of active efflux pumps (21). To date, the following five efflux systems have been described in A. baumannii: AdeABC and AdeIJK, belonging to the resistance-nodulation-cell division family and conferring resistance to a wide spectrum of antimicrobial agents (3, 9); Tet(A) and Tet(B), from the major facilitator superfamily (MFS), involved in tetracycline and minocycline resistance (10, 11); and AbeM, included within the multidrug and toxic compound extrusion family, providing moderate resistance to several compounds (16).The recent publication of up to six complete A. baumannii genomes (1, 7, 15, 18), however, accounts for the identification of novel putative efflux systems that may add to those already described in the literature, although experimental proof of their role in antimicrobial resistance is still needed. In this work, we have used homology search-based methods to identify an open reading frame (initially named orf3) encoding a putative efflux pump ortholog present in all fully sequenced A. baumannii strains (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"ABO13543.2","term_id":"193078527","term_text":"ABO13543.2"}}ABO13543.2). Its deduced amino acid sequence suggests that it consists of 409 residues and contains 12 transmembrane domains (PredictProtein server) (12, 13). It also showed sequence similarity (41% identity and 61% similarity) to the Escherichia coli MdfA protein (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"CAA69997.1","term_id":"1619595","term_text":"CAA69997.1"}}CAA69997.1), which has been previously described as an MFS efflux pump conferring resistance mainly to ciprofloxacin and chloramphenicol, among others (4). Orf3 was 99% identical among all sequenced Acinetobacter strains and it was also detected by PCR analysis in 82 out of 82 A. baumannii clonally different clinical isolates from our culture collection.To demonstrate the involvement of Orf3 in MDR, the orf3 gene from A. baumannii strain ATCC 19606 was cloned into the SmaI site of the suicide vector pEX100T (14) and disrupted by the insertion of a Km^r cassette obtained from pUC4K (19). Insertion was at nucleotide position 457 after introducing a BamHI site using a two-step nested PCR mutagenesis. The resulting construct (designated pJV102) was mobilized from the E. coli strain S-17λpir to A. baumannii strain ATCC 19606 Rif^r in order to knock out its cognate orf3 gene by allelic replacement. Exconjugants were selected on LB agar plates containing 75 μg/ml rifampin (rifampicin) and 50 μg/ml kanamycin, and cells growing on these plates were further streaked on LB agar containing 50 μg/ml kanamycin and 2% sucrose, to ensure the loss of pJV102. orf3 disruption within the resulting strain, designated JVAB01, was verified by PCR analysis.Etest susceptibility assays to quinolones, tetracyclines, aminoglycosides, and imipenem (Table ​(Table1)1) showed identical MICs between ATCC 19606 and JVAB01, but a dramatic drop in chloramphenicol resistance was detected for the Orf3 mutant strain (a MIC value of >256 μg/ml for ATCC 19606 compared to 2 μg/ml for JVAB01).

TABLE 1.

MICs of antimicrobial agents in the investigated A. baumannii strains

Association of Class 1 and 2 Integrons with Multidrug-Resistant Acinetobacter baumannii International Clones and Acinetobacter nosocomialis Isolates

The Acinetobacter baumannii clonal complex 113/79 (CC113/79) and class 2 integrons predominate in Latin America; a relationship between these characteristics was explored. The presence of integrases was determined in successive hospital Acinetobacter isolates (163 A. baumannii isolates and 72 Acinetobacter nosocomialis isolates). Most isolates had integrons, but class 1 and 2 integrons were present significantly more often in CC109/1 and CC113/79, respectively. The high prevalence of CC113/79 in Latin America may account for the predominance of class 2 integrons.     

Resistance to Colistin in Acinetobacter baumannii Associated with Mutations in the PmrAB Two-Component System

The mechanism of colistin resistance (Col^r) in Acinetobacter baumannii was studied by selecting in vitro Col^r derivatives of the multidrug-resistant A. baumannii isolate AB0057 and the drug-susceptible strain ATCC 17978, using escalating concentrations of colistin in liquid culture. DNA sequencing identified mutations in genes encoding the two-component system proteins PmrA and/or PmrB in each strain and in a Col^r clinical isolate. A colistin-susceptible revertant of one Col^r mutant strain, obtained following serial passage in the absence of colistin selection, carried a partial deletion of pmrB. Growth of AB0057 and ATCC 17978 at pH 5.5 increased the colistin MIC and conferred protection from killing by colistin in a 1-hour survival assay. Growth in ferric chloride [Fe(III)] conferred a small protective effect. Expression of pmrA was increased in Col^r mutants, but not at a low pH, suggesting that additional regulatory factors remain to be discovered.Among gram-negative pathogens that are reported as “multidrug resistant” (MDR), Acinetobacter baumannii is rapidly becoming a focus of significant attention (1, 7, 25, 32, 38, 39, 46, 51). In intensive care units, up to 30% of A. baumannii clinical isolates are resistant to at least three classes of antibiotics, often including fluoroquinolones and carbapenems (25).The emergence of MDR gram-negative pathogens, including A. baumannii, has prompted increased reliance on the cationic peptide antibiotic colistin (12). Regrettably, increasing colistin use has led to the discovery of resistant strains (10, 11, 22, 26). For example, in a recent study, 12% of carbapenemase-producing Enterobacteriaceae were found to be colistin resistant (Col^r) (6). Although still uncommon, A. baumannii isolates resistant to all available antimicrobial agents have been reported (26, 45) and are of enormous concern, given their potential to spread in the critical care environment.Colistin and other polymyxins are cyclic cationic peptides produced by the soil bacterium Bacillus polymyxa that act by disrupting the negatively charged outer membranes of gram-negative bacteria (37, 50). The following three distinct mechanisms that give rise to colistin resistance are known: (i) specific modification of the lipid A component of the outer membrane lipopolysaccharide, resulting in a reduction of the net negative charge of the outer membrane; (ii) proteolytic cleavage of the drug; and (iii) activation of a broad-spectrum efflux pump (13, 14, 49). The mechanism of colistin resistance in Acinetobacter spp. is not yet known. Heteroresistance to colistin in A. baumannii has been described (17, 24), but it is uncertain whether the basis for this resistance is the presence of a genetically distinct population of cells or whether variation in the regulatory program among genetically identical cells may be sufficient for the expression of resistance.In Salmonella enterica, the two-component signaling systems PmrAB and PhoPQ are involved in sensing environmental pH, Fe³⁺, and Mg²⁺ levels, leading to altered expression of a set of genes involved in lipid A modification (14, 43, 53). A small adapter protein, PmrD, serves as an interface between the two-component systems by stabilizing the activated form of PmrA in S. enterica (19), but other mechanisms of coordinated regulation are described for other species (52). Mutations causing constitutive activation of PmrA and PmrB are associated with colistin resistance (31, 33). Interestingly, the phoPQ and pmrD genes do not appear to be present in Acinetobacter spp., based on computational analysis of the genome sequences (2).PmrA-regulated resistance to colistin in S. enterica and P. aeruginosa results from modification of lipid A with 4-deoxy-aminoarabinose (Ara4N) or phosphoethanolamine via activation of ugd, the pmrF (or pbgP) operon, and pmrC, which encode UDP-glucose dehydrogenase (the first step in Ara4N biosynthesis), Ara4N biosynthetic enzymes, and lipid A phosphoethanolamine transferase, respectively (8, 15, 21, 41, 48). The Ara4N biosynthesis and attachment genes are not present in A. baumannii or Neisseria meningitidis (36, 47). N. meningitidis is intrinsically resistant to polymyxins, demonstrating that Ara4N modification of lipid A is not required for resistance. Mutations in the pmrC ortholog lptA, encoding the lipid A phosphoethanolamine transferase, reduce colistin resistance in N. meningitidis, suggesting that this modification alone may be sufficient for conferring colistin resistance (49). Here we show that the PmrAB system is involved in regulating colistin resistance in A. baumannii by identification of mutations in resistant isolates that exhibit constitutive expression of pmrA.     

Antimicrobial Activity of Nanoemulsion in Combination with Cetylpyridinium Chloride in Multidrug-Resistant Acinetobacter baumannii

Acinetobacter baumannii has emerged as a serious problematic pathogen due to the ever-increasing presence of antibiotic resistance, demonstrating a need for novel, broad-spectrum antimicrobial therapeutic options. Antimicrobial nanoemulsions are emulsified mixtures of detergent, oil, and water (droplet size, 100 to 800 nm) which have broad antimicrobial activity against bacteria, enveloped viruses, and fungi. Here, we screened the antimicrobial activities of five nanoemulsion preparations against four Acinetobacter baumannii isolates to identify the most suitable preparation for further evaluation. Among them, N5, which contains 10% (vol/vol) Triton X-100, 25% (vol/vol) soybean oil, and 1% (wt/vol) cetylpyridinium chloride (CPC), showed the best efficacy against A. baumannii in both its planktonic and biofilm forms and was selected for further study. Our data demonstrate that, while the killing of planktonic forms of A. baumannii was due to the 1% CPC component of our nanoemulsions, the breakdown of biofilms was achieved via the emulsified oil and detergent fractions. Furthermore, we documented the effect of ethanol and NaCl in combination with N5 on planktonic A. baumannii. In killing curves of N5 combined with other agents (ethanol or NaCl), a synergistic effect of a ≥2-log decrease in CFU/ml was observed. The antibiofilm activity of N5 was confirmed via a cell proliferation test and scanning electron microscopy. The effects of exposure to severe environmental conditions, which simulates the field conditions in Iraq and Afghanistan, were evaluated, and this exposure did not affect the overall antimicrobial activity of N5. These studies lay a solid foundation for the utilization of nanoemulsions against the antibiotic-resistant forms of A. baumannii.     

A Study of Fifty-eight Patients with Renal Scarring Associated with Urinary Tract Infection

Fifty-eight patients, three male and 55 female, with radiologicallyapparent renal scarring together with urinary tract infectionhave been followed for periods from five to 13 years. Controlof urinary infection has been attempted by antibacterial treatmentand prophylaxis, and radiological appearances, renal functionand blood pressure have been monitored. Urinary infection waseliminated in 50 patients (86 per cent), all of whom becamesymptom-free. Evidence of new renal scarring was not seen, butthree patients showed contraction of kidney size and three developedradio-opaque renal calculi. Vesico-ureteric reflux was demonstratedin 20 patients; surgical re-implantation of ureters was carriedout in four patients and the indications for this operationare discussed. Of the 12 patients who had a raised serum creatinineat some time during the study only three showed progressivedecline in renal function; the serum creatinine returned tonormal in seven patients when infection was controlled. Hypertensionwas more common in patients with bilateral than in those withunilateral scarring, and all patients with evidence of impairedfunction at any time developed hypertension. We conclude thatmost patients with scarred kidneys and urinary infection canbe rendered symptom-free by control of infection, and that thereis some evidence that renal function can be preserved in thisway.