Characterization of Amino Acid Substitutions in the Two-Component Regulatory System AdeRS Identified in Multidrug-Resistant Acinetobacter baumannii.

In Acinetobacter baumannii, resistance-nodulation-cell division (RND)-type efflux is a resistance mechanism of great importance since it contributes to reduced susceptibility to multiple antimicrobial compounds. Some mutations within the genes encoding the two-component regulatory system AdeRS appear to play a major role in increased expression of the RND efflux pump AdeABC and, consequently, in reduced antimicrobial susceptibility, as they are commonly observed in multidrug-resistant (MDR) A. baumannii. In the present study, the impact of frequently identified amino acid substitutions, namely, D21V and D26N in AdeR and T156M in AdeS, on adeB expression, efflux activity, and antimicrobial susceptibility was investigated. Reverse transcription-quantitative PCR (qRT-PCR) studies revealed significantly increased adeB expression caused by D26N (AdeR) and T156M (AdeS). In addition, accumulation assays have shown that these mutations induce increased efflux activity. Subsequently, antimicrobial susceptibility testing via agar dilution and broth microdilution confirmed the importance of these substitutions for the MDR phenotype, as the MICs for various antimicrobials of different classes were increased. In contrast, the amino acid substitution D21V in AdeR did not lead to increased adeB expression and did not reduce antimicrobial susceptibility. This study demonstrates the impact of the D26N (AdeR) and T156M (AdeS) amino acid substitutions, highlighting that these regulators represent promising targets for interfering with efflux activity to restore antimicrobial susceptibility. IMPORTANCE The active efflux of antimicrobials by bacteria can lead to antimicrobial resistance and persistence and can affect multiple different classes of antimicrobials. Efflux pumps are tightly regulated, and their overexpression can be mediated by changes in their regulators. Identifying these changes is one step in the direction of resistance prediction, but it also opens the possibility of targeting efflux pump regulation as a strategy to overcome antimicrobial resistance. Here, we have investigated commonly found changes in the regulators of the main efflux pumps in Acinetobacter baumannii.

Outbreak of Pseudomonas aeruginosa infections after CT-guided spinal injections.

BACKGROUND: Meningitis and spinal infections with Gram-negative bacteria after local injections for treatment of chronic back pain are rare. This study investigated an outbreak of Pseudomonas aeruginosa infections following computed tomography (CT)-guided spinal injections (SI). METHODS: A case was defined as a spinal infection or meningitis with P. aeruginosa after SI between 10th January and 1st March 2019 in the same outpatient clinic. Patients without microbiological evidence of P. aeruginosa but with a favourable response to antimicrobial therapy active against P. aeruginosa were defined as probable cases. FINDINGS: Twenty-eight of 297 patients receiving CT-guided SI during the study period developed meningitis or spinal infections. Medical records were available for 19 patients. In 15 patients, there was microbiological evidence of P. aeruginosa, and four patients were defined as probable cases. Two of 19 patients developed meningitis, while the remaining 17 patients developed spinal infections. The median time from SI to hospital admission was 8 days (interquartile range 2-23 days). Patients mainly presented with back pain (N=18; 95%), and rarely developed fever (N=3; 16%). Most patients required surgery (N=16; 84%). Seven patients (37%) relapsed and one patient died. Although the source of infection was not identified microbiologically, documented failures in asepsis when performing SI probably contributed to these infections. CONCLUSIONS: SI is generally considered safe, but non-adherence to asepsis can lead to deleterious effects. Spinal infections caused by P. aeruginosa are difficult to treat and have a high relapse rate.

Comparison of the Acinetobacter baumannii Reference Strains ATCC 17978 and ATCC 19606 in Antimicrobial Resistance Mediated by the AdeABC Efflux Pump.

The Acinetobacter baumannii RND efflux pump AdeABC is regulated by the 2-component regulator AdeRS. In this study, we compared the regulation and expression of AdeABC of the reference strains ATCC 17978 and ATCC 19606. A clearly stronger efflux activity was demonstrated for ATCC 19606. An amino acid substitution at residue 172 of adeS was identified as a potential cause for differential expression of the pump. Therefore, we recommend caution with exclusively using single reference strains for research.

High incidence of pandrug-resistant Acinetobacter baumannii isolates collected from patients with ventilator-associated pneumonia in Greece, Italy and Spain as part of the MagicBullet clinical trial.

OBJECTIVES: To investigate the molecular epidemiology, antimicrobial susceptibility and carbapenem resistance determinants of Acinetobacter baumannii isolates from respiratory tract samples of patients diagnosed with ventilator-associated pneumonia (VAP) who were enrolled in the MagicBullet clinical trial. METHODS: A. baumannii isolates were prospectively cultured from respiratory tract samples from 65 patients from 15 hospitals in Greece, Italy and Spain. Susceptibility testing was performed by broth microdilution. Carbapenem resistance determinants were identified by PCR and sequencing. Molecular epidemiology was investigated using rep-PCR (DiversiLab) and international clones (IC) were identified using our in-house database. RESULTS: Of 65 isolates, all but two isolates (97%) were resistant to imipenem and these were always associated with an acquired carbapenemase, OXA-23 (80%), OXA-40 (4.6%), OXA-58 (1.5%) or OXA-23/58 (1.5%). Resistance to colistin was 47.7%. Twenty-two isolates were XDR, and 20 isolates were pandrug-resistant (PDR). The majority of isolates clustered with IC2 (n = 54) with one major subtype comprising isolates from 12 hospitals in the three countries, which included 19 XDR and 16 PDR isolates. CONCLUSIONS: Carbapenem resistance rates were very high in A. baumannii recovered from patients with VAP. Almost half of the isolates were colistin resistant, and 42 (64.6%) isolates were XDR or PDR. Rep-PCR confirmed IC2 is the predominant clonal lineage in Europe and suggests the presence of an epidemic XDR/PDR A. baumannii clone that has spread in Greece, Italy and Spain. These data highlight the difficulty in empirical treatment of patients with A. baumannii VAP in centres with a high prevalence of carbapenem-resistant A. baumannii.

MALDI-TOF/MS identification of species from the Acinetobacter baumannii (Ab) group revisited: inclusion of the novel A. seifertii and A. dijkshoorniae species.

OBJECTIVES: Rapid identification of Acinetobacter species is critical as members of the A. baumannii (Ab) group differ in antibiotic susceptibility and clinical outcomes. A. baumannii, A. pittii, and A. nosocomialis can be identified by MALDI-TOF/MS, while the novel species A. seifertii and A. dijkshoorniae cannot. Low identification rates for A. nosocomialis also have been reported. We evaluated the use of MALDI-TOF/MS to identify isolates of A. seifertii and A. dijkshoorniae and revisited the identification of A. nosocomialis to update the Bruker taxonomy database. METHODS: Species characterization was performed by rpoB-clustering and MLSA. MALDI-TOF/MS spectra were recovered from formic acid/acetonitrile bacterial extracts overlaid with α-cyano-4-hydroxy-cinnamic acid matrix on a MicroflexLT in linear positive mode and 2000-20 000 m/z range mass. Spectra were examined with the ClinProTools v2.2 software. Mean spectra (MSP) were created with the BioTyper software. RESULTS: Seventy-eight Acinetobacter isolates representative of the Ab group were used to calculate the average spectra/species and generate pattern recognition models. Species-specific peaks were identified for all species, and MSPs derived from three A. seifertii, two A. dijkshoorniae, and two A. nosocomialis strains were added to the Bruker taxonomy database, allowing successful identification of all isolates using spectra from either bacterial extracts or direct colonies, resulting in a positive predictive value (PPV) of 99.6% (777/780) and 96.8% (302/312), respectively. CONCLUSIONS: The use of post-processing data software identified statistically significant species-specific peaks to generate reference signatures for rapid accurate identification of species within the Ab group, providing relevant information for the clinical management of Acinetobacter infections.

Draft Genome Sequences of 11 Clinical Isolates of Acinetobacter baumannii.

The development of multidrug-resistantAcinetobacter baumanniiis of serious concern in the hospital setting. Here, we report draft genome sequences of 11A. baumanniiisolates that were isolated from a single patient over a 65-day period, during which time the isolates exhibited increased antimicrobial resistance.

Outbreak of carbapenem-resistant Acinetobacter baumannii in the intensive care unit: a multi-level strategic management approach.

An outbreak of carbapenem-resistant Acinetobacter baumannii (CRAb) occurred in an interdisciplinary intensive care unit, affecting 10 patients. Within hours of recognition of the spread of CRAb an intervention team was instituted for collection of available data, decision-making, communication and monitoring of all interventions performed, including cohorting, temporary stop of admissions, staff education, and enforcement of infection control measures. An area was defined for cohortation of patients colonized with CRAb, with a separate nursing team and a second set of mobile equipment. New transmissions were no longer observed after only four days into the institution of enhanced infection control measures.

Molecular epidemiology of Acinetobacter baumannii and Acinetobacter nosocomialis in Germany over a 5-year period (2005-2009).

To investigate the species distribution within the Acinetobacter calcoaceticus-Acinetobacter baumannii complex and the molecular epidemiology of A. baumannii and Acinetobacter nosocomialis, 376 Acinetobacter isolates were collected prospectively from hospitalized patients at 15 medical centres in Germany during three surveillance studies conducted over a 5-year period. Species identification was performed by molecular methods. Imipenem minimum inhibitory concentrations (MIC) were determined by broth microdilution. The prevalence of the most common carbapenemase-encoding genes was investigated by oxacillinase (OXA) -multiplex polymerase chain reaction (PCR). The molecular epidemiology was investigated by repetitive sequence-based PCR (rep-PCR; DiversiLab™). Acinetobacter pittii was the most prevalent Acinetobacter species (n = 193), followed by A. baumannii (n = 140), A. calcoaceticus (n = 10) and A. nosocomialis (n = 8). The majority of A. baumannii was represented by sporadic isolates (n = 70, 50%) that showed unique rep-PCR patterns, 25 isolates (18%) clustered with one or two other isolates, and only 45 isolates (32%) belonged to one of the previously described international clonal lineages. The most prevalent clonal lineage was international clone (IC) 2 (n = 34) and IC 1 (n = 6). According to CLSI, 25 A. baumannii isolates were non-susceptible to imipenem (MIC ≥ 8 mg/L), all of which produced an OXA-58-like or OXA-23-like carbapenemase. The rate of imipenem susceptibility among A. baumannii isolates decreased from 96% in 2005 to 76% in 2009. All other Acinetobacter isolates were susceptible to imipenem. The population structure of carbapenem-susceptible A. baumannii in Germany is highly diverse. Imipenem non-susceptibility was strongly associated with the clonal lineages IC 2 and IC 1. These data underscore the high clonality of carbapenem-resistant A. baumannii isolates.

Control of an outbreak of Acinetobacter baumannii infections using vaporized hydrogen peroxide.

BACKGROUND: Multidrug-resistant Acinetobacter baumannii (MRAB) is a serious nosocomial pathogen characterized by its survival on inanimate surfaces for long periods, making control of outbreaks difficult. AIM: To analyse two hospital outbreaks caused by MRAB, determine their epidemiology, carbapenem-resistance mechanisms and assess the effectiveness of surface disinfection by vaporized hydrogen peroxide (VHP). METHODS: MRAB strains were isolated from patients in two intensive care units (ICUs). Antimicrobial susceptibility testing was performed by E-test. Polymerase chain reaction (PCR) was used to detect the presence of the most common A. baumannii carbapenemases. Epidemiological typing was performed by rep-PCR (DiversiLab) and pulsed-field gel electrophoresis. VHP was used to decontaminate the affected ICUs. FINDINGS: MRAB was isolated from 28 patients between January 2009 and September 2010. All isolates were resistant to ciprofloxacin and gentamicin. Twenty-one were also resistant to carbapenems. Carbapenem resistance was associated primarily with the acquired OXA-23-like enzyme. Genotyping revealed three clones; the predominant clone corresponded to the international clone (IC) 2. Typing of the isolates pointed to a multifocal outbreak without a single source of infection, with horizontal spread of the dominating clone among ICU patients. A combination of rigorous infection control measures including strict isolation, education of staff, hand hygiene and surface decontamination using VHP halted the outbreak. CONCLUSION: The results of this study confirm the importance of rigorous infection prevention and control measures, combined with VHP decontamination in controlling an outbreak of MRAB.

Molecular epidemiology of clinical Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates using a multilocus sequencing typing scheme.

To further expand the limited multilocus sequence typing (MLST) database for Acinetobacter baumannii, 53 clinical isolates from various outbreaks in Europe and the USA, collected between 1991 and 2004, plus the A. baumannii reference strain ATCC 19606(T) and 20 clinical Acinetobacter genomic species 13TU isolates from the same period, were analyzed using a new MLST scheme based on fragments of the gltA, gyrB, gdhB, recA, cpn60, gpi and rpoD genes. Data were compared with typing results generated using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD)-PCR. In total, 50 sequence types (STs) were distinguished among the A. baumannii isolates investigated, and the MLST data were in high concordance with the PFGE and RAPD-PCR results. Only five clonal complexes were identified by eBURST analysis, including the 21 STs listed in a previous study, suggesting high diversity among the A. baumannii isolates. With one exception, there was no relatedness among isolates from outbreaks in different countries (Europe) or regions (USA). No intercontinental spread was revealed. Acinetobacter genomic species 13TU isolates could also be analyzed using the A. baumannii MLST scheme (18 different STs) and could be distinguished from A. baumannii isolates according to characteristic sequences. It was concluded that the MLST scheme provides a high level of resolution and is a promising tool for studying the epidemiology of A. baumannii and Acinetobacter genomic species 13TU.

Clonal spread of carbapenem-resistant OXA-23-positive Acinetobacter baumannii in a Bulgarian university hospital.

From October 1999 to September 2006, 29 carbapenem-resistant isolates of Acinetobacter baumannii were collected consecutively from patients hospitalized in different wards of the University Hospital in Pleven, Bulgaria. The bla(OXA-23) gene, associated with the upstream-located ISAba1, was identified as the mechanism responsible for carbapenem resistance in all isolates. The isolates belonged to two different clonal groups, indicating a sustained hospital outbreak. This study demonstrates both the epidemic potential of carbapenem-resistant A. baumannii and its longevity in the hospital environment.

A PCR-based method to differentiate between Acinetobacter baumannii and Acinetobacter genomic species 13TU.

A new PCR-based method that exploits differences in gyrB gene sequences was developed to distinguish between Acinetobacter baumannii and Acinetobacter genomic sp. 13TU. Among 118 clinical and reference Acinetobacter strains, 102 of which were previously speciated by amplified rDNA restriction analysis as belonging to the Acinetobacter calcoaceticus-A. baumannii complex, the method correctly identified 31 A. baumannii and 54 Acinetobacter genomic sp. 13TU isolates to the species level. The method was rapid, specific and easy to interpret.

Mutations in GyrA, ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa.

The target enzymes GyrA and ParC and two efflux pump regulatory genes mexR and nfxB were analysed to determine changes associated with fluoroquinolone resistance in Pseudomonas aeruginosa. Both low- and high-level ciprofloxacin resistance was associated with a Thr-83Ile substitution in GyrA. A ParC Ser-80Leu substitution was found in highly resistant isolates in tandem with the Thr-83Ile substitution in GyrA. Mutations in the efflux regulatory genes were associated with resistance only when in tandem with a mutation in GyrA or ParC. These data show that the main mechanism of fluoroquinolone resistance in P. aeruginosa is mediated primarily through mutations in GyrA, and that mutations in ParC and the efflux regulatory genes are secondary.

Fluoroquinolones: structure and target sites.

The quinolones are a potent group of drugs that target the essential bacterial enzymes DNA gyrase and topoisomerase IV. DNA gyrase is the primary target of Gram negative organisms however, it is topoisomerase IV that is the primary target of Gram positive organisms. Within these enzymes is a highly conserved region centered round the active site where resistance mutations occur. These mutations are almost always identical, irrespective of organism. In spite of the homology of this region, amino acid sequence analysis shows that there are defined differences between the Gram groups, particularly in topoisomerase IV, and it is speculated that herein lies the origin of target preference. Since the first quinolone nalidixic acid was developed, the quinolones have undergone structural modifications, in particular the addition of a fluorine at position 6, to produce the fluoroquinolones. This has seen their potency and pharmakokinetic profile greatly increase. In vitro selection of resistance mutations has allowed the observation of how resistance is acquired and some of the modifications in newer fluoroquinolones have resulted in the shift of primary target from topoisomerase IV to gyrase with Gram positives. Curiously, purified topoisomerase IV is still more sensitive even if gyrase is the primary target. Gyrase remains the primary target for Gram negatives.

Activity of quinolones against gram-positive cocci: mechanisms of drug action and bacterial resistance.

The quinolones are a potent class of antimicrobial agents that target two essential enzymes of bacterial cells: DNA gyrase and topoisomerase IV. Resistance is mediated chiefly through stepwise mutations in the genes that encode these enzymes, leading to alterations of the target site. These mutations occur in an area called the "quinolone resistance determining region". In gram-positive organisms, mutations occur more often in topoisomerase IV than in DNA gyrase. This target preference appears to depend upon two factors: the species of organism and the selecting drug. Resistance can be enhanced by a decrease in intracellular drug concentration, which is mediated through efflux pumps. The newer generation of fluoroquinolones and non-fluorinated quinolones exhibits enhanced activity against gram-positive organisms compared to the older members of this drug class, although development of resistance to these drugs has been demonstrated in vitro. This review gives a chronological perspective of the literature on the action of DNA gyrase and topoisomerase IV and the mechanisms of resistance to quinolones in staphylococci, streptococci and enterococci.

Bactericidal and bacteriostatic activity of gemifloxacin against Acinetobacter spp. in vitro.

This study compared the in vitro bacteriostatic activity of gemifloxacin (SB-265805) and a panel of test antimicrobial agents against 100 clinical isolates of Acinetobacter spp. (47 Acinetobacter baumannii, 18 Acinetobacter anitratus, 18 Acinetobacter lwoffii, 13 Acinetobacter calcoaceticus and four other Acinetobacter spp.). Gemifloxacin (MIC(50/90) 0.06/16 mg/L) was more than eight-fold more potent than ciprofloxacin (0.5/>128 mg/L), two- to eight-fold more potent than grepafloxacin, moxifloxacin, levofloxacin, ofloxacin and gatifloxacin, and of similar potency to trovafloxacin and sparfloxacin. Cross-resistance was seen only within the quinolone group and did not extend to non-quinolone antimicrobials. The bactericidal activities of gemifloxacin and the six comparator quinolones were investigated by dose-response and time-kill studies against A. baumannii ATCC 19606 at their optimum bactericidal concentration (OBC) and at 4 x MIC. At the OBC there was no significant difference between the quinolones, but at 4 x MIC gemifloxacin showed superior activity, reducing the viable count by almost 2 log(10) in 30 min compared with a 1 log(10) reduction seen with the other drugs. This enhanced killing extended over 24 h, reducing cell numbers by >4 log(10). These data suggest that gemifloxacin has the potential to be of therapeutic value in the treatment of infection by Acinetobacter spp.

Reasons, health behaviors, and outcomes of no prenatal care: research that changed practice.

Changes in prenatal care practices resulted from a pilot study with 12 urban New Mexican women who received no prenatal care. The women were interviewed regarding their reasons for not receiving care during pregnancy, health behaviors, and perceived neonatal outcomes. Data on actual neonatal outcomes were taken from the medical record. Maternal reasons for no prenatal care were socio-demographic, system-related, attitudinal, and outside forces of job and childcare. To ensure a healthy baby, the women made changes in their nutrition, self-care activities, substance use, sleep, and exercise activities. All of the women perceived they had a healthy baby. Yet 61% of the neonates had complications and 45% were low birth weight. The research findings were used to develop a care management program that included case management and utilization management.