Prevalence of Acinetobacter baumannii and Candida auris in Patients Receiving Mechanical Ventilation.

Importance: To date, only 1 statewide prevalence survey has been performed for Acinetobacter baumannii (2009) in the US, and no statewide prevalence survey has been performed for Candida auris, making the current burden of these emerging pathogens unknown. Objective: To determine the prevalence of A baumannii and C auris among patients receiving mechanical ventilation in Maryland. Design, Setting, and Participants: The Maryland Multi-Drug Resistant Organism Prevention Collaborative performed a statewide cross-sectional point prevalence of patients receiving mechanical ventilation admitted to acute care hospitals (n = 33) and long-term care facilities (n = 18) between March 7, 2023, and June 8, 2023. Surveillance cultures (sputum, perianal, arm/leg, and axilla/groin) were obtained from all patients receiving mechanical ventilation. Sputum, perianal, and arm/leg cultures were tested for A baumannii and antibiotic susceptibility testing was performed. Axilla/groin cultures were tested by polymerase chain reaction for C auris. Main Outcomes and Measures: Prevalence of A baumannii, carbapenem-resistant A baumannii (CRAB), and C auris. Prevalence was stratified by type of facility. Results: All 51 eligible health care facilities (100%) participated in the survey. A total of 482 patients receiving mechanical ventilation were screened for A baumannii and 470 were screened for C auris. Among the 482 patients who had samples collected, 30.7% (148/482) grew A baumannii, 88 of the 148 (59.5%) of these A baumannii were CRAB, and C auris was identified in 31 of 470 (6.6%). Patients in long-term care facilities were more likely to be colonized with A baumannii (relative risk [RR], 7.66 [95% CI, 5.11-11.50], P < .001), CRAB (RR, 5.48 [95% CI, 3.38-8.91], P < .001), and C auris (RR, 1.97 [95% CI, 0.99-3.92], P = .05) compared with patients in acute care hospitals. Nine patients (29.0%) with cultures positive for C auris were previously unreported to the Maryland Department of Health. Conclusions: A baumannii, carbapenem-resistant A baumannii, and C auris were common among patients receiving mechanical ventilation in both acute care hospitals and long-term care facilities. Both pathogens were significantly more common in long-term care facilities than in acute care hospitals. Patients receiving mechanical ventilation in long-term care facilities are a high-risk population for emerging pathogens, and surveillance and prevention efforts should be targeted to these facilities.

A SERS bioassay based on vancomycin-modified PEI-interlayered nanocomposite and aptamer-functionalized SERS tags for synchronous detection of Acinetobacter baumannii and Klebsiella pneumoniae.

Klebsiella pneumoniae (KP) and Acinetobacter baumannii (AB) are two important gram-negative bacteria that cause pneumonia and have been recently known to be associated with food. The rapid detection of these pathogens in food is important to minimize their colonization of the gut and stop new threats of the disease from spreading across the food chain. Herein, a double-edged sword aptasensor was developed for the synchronous detection of KP and AB in food and clinical samples. A highly sensitive, selective, specific, and synchronous detection of the target bacteria was achieved, and the limit of detection (LOD) was 10 cells/mL with a liner range of 50 to 105 cells/mL. The total assay time was 1.5 h. This study does not only provide a new tool for the detection of the target bacteria, but also serves as a promising tool for food safety and pneumonia diagnosis.

The evolution of carbapenem resistance determinants and major epidemiological lineages among carbapenem-resistant Acinetobacter baumannii isolates in Germany, 2010-2019.

The aim of this study was to investigate and compare the molecular epidemiology and carbapenem resistance determinants in clinical Acinetobacter baumannii isolates collected during four multicentre surveillance studies conducted by the Paul-Ehrlich-Society for Infection Therapy. Isolates were collected prospectively from hospital in-patients at 17 medical centres in Germany over four periods of three- to six-months starting in October of each of 2010, 2013, 2016 and 2019. Species identification was performed by MALDI-TOF, gyrB multiplex polymerase chain reaction (PCR), and detection of the intrinsic blaOXA-51-like gene. Minimum inhibitory concentrations were determined by broth microdilution. The prevalence of carbapenemase-encoding genes was investigated by OXA-multiplex PCR and whole-genome sequencing. Molecular epidemiology was examined by rep-PCR and core-genome multi-locus sequence typing. A total of 302 A. baumannii isolates were collected. Resistance to imipenem and/or meropenem was detected in 58 isolates (19.2%) from 14 centres. The proportion of carbapenem-resistant isolates increased from 21.3% in 2010 to 33.3% in 2013, and then decreased to 13.8% in 2016 and 12.3% in 2019. Forty-six of these isolates were associated with the international clonal lineage IC2 and five with IC1. The most prevalent carbapenemase gene detected was blaOXA-23-like (n=51). Further carbapenem-resistance determinants were blaOXA-40-like (n=1), blaOXA-58-like (n=3) and blaNDM-1 (n=2). In one isolate, ISAba1 was detected upstream of blaOXA-51-like. In conclusion, IC2 was the most prevalent clonal lineage detected in this study. Interestingly, in Germany, carbapenem resistance seems to have decreased in A. baumannii between 2013 and 2019.

Factors influencing environmental sampling recovery of healthcare pathogens from non-porous surfaces with cellulose sponges.

Results from sampling healthcare surfaces for pathogens are difficult to interpret without understanding the factors that influence pathogen detection. We investigated the recovery of four healthcare-associated pathogens from three common surface materials, and how a body fluid simulant (artificial test soil, ATS), deposition method, and contamination levels influence the percent of organisms recovered (%R). Known quantities of carbapenemase-producing KPC+ Klebsiella pneumoniae (KPC), Acinetobacter baumannii, vancomycin-resistant Enterococcus faecalis, and Clostridioides difficile spores (CD) were suspended in Butterfield's buffer or ATS, deposited on 323cm2 steel, plastic, and laminate surfaces, allowed to dry 1h, then sampled with a cellulose sponge wipe. Bacteria were eluted, cultured, CFU counted and %R determined relative to the inoculum. The %R varied by organism, from <1% (KPC) to almost 60% (CD) and was more dependent upon the organism's characteristics and presence of ATS than on surface type. KPC persistence as determined by culture also declined by >1 log10 within the 60 min drying time. For all organisms, the %R was significantly greater if suspended in ATS than if suspended in Butterfield's buffer (p<0.05), and for most organisms the %R was not significantly different when sampled from any of the three surfaces. Organisms deposited in multiple droplets were recovered at equal or higher %R than if spread evenly on the surface. This work assists in interpreting data collected while investigating a healthcare infection outbreak or while conducting infection intervention studies.

Genome diversity of domesticated Acinetobacter baumannii ATCC 19606T strains.

Acinetobacter baumannii has emerged as an important opportunistic pathogen worldwide, being responsible for large outbreaks for nosocomial infections, primarily in intensive care units. A. baumannii ATCC 19606T is the species type strain, and a reference organism in many laboratories due to its low virulence, amenability to genetic manipulation and extensive antibiotic susceptibility. We wondered if frequent propagation of A. baumannii ATCC 19606T in different laboratories may have driven micro- and macro-evolutionary events that could determine inter-laboratory differences of genome-based data. By combining Illumina MiSeq, MinION and Sanger technologies, we generated a high-quality whole-genome sequence of A. baumannii ATCC 19606T, then performed a comparative genome analysis between A. baumannii ATCC 19606T strains from several research laboratories and a reference collection. Differences between publicly available ATCC 19606T genome sequences were observed, including SNPs, macro- and micro-deletions, and the uneven presence of a 52 kb prophage belonging to genus Vieuvirus. Two plasmids, pMAC and p1ATCC19606, were invariably detected in all tested strains. The presence of a putative replicase, a replication origin containing four 22-mer direct repeats, and a toxin-antitoxin system implicated in plasmid stability were predicted by in silico analysis of p1ATCC19606, and experimentally confirmed. This work refines the sequence, structure and functional annotation of the A. baumannii ATCC 19606T genome, and highlights some remarkable differences between domesticated strains, likely resulting from genetic drift.

Immunological Evaluation of Individuals Infected with Acinetobacter baumannii.

Acinetobacter baumannii (A. baumannii) is a spherical rod-shaped Gram-negative non-lactose fermenting (Coccobacilli, Aerobic bacteria) bacteria. It is a member of the Moraxellacea family. A. baumannii is a pathogenic, opportunistic organism that infects humans in society and hospitals. In particular, patients with immune system defects are at risk, especially those with burn infections and those hospitalized in intensive care (ICU). It plays a vital role in many illnesses, including septicemia, pneumonia, meningitis, soft tissues, skin infection, endocarditis, and urinary tract infection (UTI). The current study included immunological evaluation of infection with A. baumannii. In the current study, 150 blood samples were obtained as follows: 100 blood samples were collected from infected individuals with A. baumannii admitted to hospitals in Baghdad. Fifty blood samples were obtained from healthy individuals and considered as the control. 10 ml of blood samples were collected from the venous blood of the participants. A. baumannii was collected and isolated from infected patients and diagnosed by traditional methods, using different culture media (MacConkey agar, blood agar, and Chromogenetic agar) and by biochemical assays, then the bacteria diagnosis was confirmed using the VITEK 2 ID-GN cards. Microscopic examination and culture diagnosis of bacteria were conducted, and the diagnosis was confirmed by complete biochemical examinations using VITEK2 Compact System. Assessments included the serum level of IL-17A and TNF-α for hospitalized patients infected with A. baumannii. The study recorded a significant increase in the serum level of IL-17A for patients infected with A. baumannii (479.83±26.21 pg/ml) compared to control subjects (69.32±4.53 pg/ml). The recorded data showed a significant increase in the serum level of TNF-α for patients infected with A. baumannii (98.05±28.89 pg/ml) compared to control (1.40±25.12 pg/ml).

Thioredoxin-mediated alteration of protein content and cytotoxicity of Acinetobacter baumannii outer membrane vesicles.

Acinetobacter baumannii is a Gram-negative bacterium responsible for many hospital-acquired infections including ventilator-associated pneumonia and sepsis. We have previously identified A. baumannii thioredoxin A protein (TrxA) as a virulence factor with a multitude of functions including reduction of protein disulfides. TrxA plays an important role in resistance to oxidative stress facilitating host immune evasion in part by alteration of type IV pili and cell surface hydrophobicity. Other virulence factors such as outer membrane vesicles (OMV) shed by bacteria have been shown to mediate bacterial intercellular communication and modulate host immune response. To investigate whether OMVs can be modulated by TrxA, we isolated OMVs from wild type (WT) and TrxA-deficient (ΔtrxA) A. baumannii clinical isolate Ci79 and carried out a functional and proteomic comparison. Despite attenuation of ΔtrxA in a mouse challenge model, pulmonary inoculation of ΔtrxA OMVs resulted in increased lung permeability compared to WT OMVs. Furthermore, ΔtrxA OMVs induced more J774 macrophage-like cell death than WT OMVs. This ΔtrxA OMV-mediated cell death was abrogated when cells were incubated with protease-K-treated OMVs suggesting OMV proteins were responsible for cytotoxicity. We therefore compared WT and mutant OMV proteins using proteomic analysis. We observed that up-regulated and unique ΔtrxA OMV proteins consisted of many membrane bound proteins involved in small molecule transport as well as proteolytic activity. Bacterial OmpA, metalloprotease, and fimbrial protein have been shown to enhance mammalian cell apoptosis through various mechanisms. Differential packaging of these proteins in ΔtrxA OMVs may contribute to the increased cytotoxicity observed in this study.

The effect of the efflux pump inhibitor Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP) on the susceptibility to imipenem and cefepime in clinical strains of Acinetobacter baumannii.

INTRODUCTION: In the last years the rapid expansion of multidrug-resistant A. baumannii strains have become a major health problem. Efflux pumps are a group of transport proteins that contribute to the development of antibiotic resistance. The aim of this study was to evaluate the effect of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the antimicrobial action of imipenem and cefepime on clinical strains of A. baumannii. MATERIALS AND METHODS: A total of 49 non-duplicate clinical samples were collected during January through December of 2018 from patients hospitalized in the Hospital Regional Docente de Cajamarca. Of the 49 samples obtained, the confirmatory identification of A. baumannii was performed on 47 samples by molecular methods. The amplification of the blaOXA-51-like gene was carried out by polymerase chain reaction (PCR). The determination of the minimum inhibitory concentration (MIC) was calculated using the microdilution method in culture broth. The susceptibility to both antibiotics (cefepime and imipenem) was evaluated in the presence and absence of the inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). RESULTS: A total of 47 strains of A. baumannii were isolated: 97.87% (46/47) were resistant to Imipenem, 2.13% (1/47) of them were classified as intermediate and none of these strains were susceptible. On the other hand, 51.06% (24/47) of isolates were resistant to cefepime; 19.15% (9/47) intermediate and 29.79% (14/47) susceptible. We considered a significant difference in antibiotic susceptibility if the MIC changed at least 4 dilutions, after the addition of the inhibitor. In the case of CCCP in addition to imipenem, 2.1% (1/47) had a significant change of 4 or more reductions in MIC, 59.6% (28/47) achieved a change equal or less than 3 dilutions and 17.0% (8/47) did not have any change. In the case of CCCP with cefepime the percentage of strains with the significant change of MIC was 8.5% (4/47). On the other hand, 53.2% (24/47) presented a reduction equal or less than 3 dilutions and 12.8% (6/47) did not show changes. CONCLUSION: In conclusion, our results demonstrate that the use of CCCP may improve the antibiotic effect of imipenem and cefepime on clinical strains of A. baumannii. The relevance of this study is that it provides evidence that this efflux pump inhibitor may be an alternative treatment against multidrug-resistant A. baumannii.

Development and clinical validation of a droplet digital PCR assay for detecting Acinetobacter baumannii and Klebsiella pneumoniae in patients with suspected bloodstream infections.

The relatively long turnaround time and low sensitivity of traditional blood culture-based diagnosis may delay effective antibiotic therapy for patients with bloodstream infections (BSIs). A rapid and sensitive pathogen detection method is urgently required to reduce the morbidity and mortality associated with BSIs. Acinetobacter baumannii and Klebsiella pneumoniae are two major microorganisms that cause BSIs. Here we report a novel droplet digital polymerase chain reaction (ddPCR) assay that can detect A. baumannii and K. pneumoniae in blood samples within 4 h, with a specificity of 100% for each strain and a limit of detection at 0.93 copies/µl for A. baumannii and 0.27 copies/µl for K. pneumoniae. Clinical validation of 170 patients with suspected BSIs showed that compared to blood cultures that detected four (2.4%) A. baumannii cases and seven (4.1%) K. pneumoniae cases, ddPCR detected 23 (13.5%) A. baumannii cases, 26 (15.3%) K. pneumoniae cases, and four (2.4%) co-infection cases, including the 11 cases detected via blood culture. In addition, patients who tested positive via ddPCR alone (n = 42) had significantly lower serum concentrations of procalcitonin and lactate, SOFA and APACHE II scores, and 28-day mortality than those reported positive via both blood culture and ddPCR (n = 11), suggesting that patients with less severe symptoms can potentially benefit from ddPCR-based diagnosis. In conclusion, our study suggests that ddPCR represents a sensitive and rapid method for identifying causal pathogens in blood samples and guiding treatment decisions in the early stages of BSIs.

Mouse pneumonia model by Acinetobacter baumannii multidrug resistant strains: Comparison between intranasal inoculation, intratracheal instillation and oropharyngeal aspiration techniques.

Infectious pneumonia induced by multidrug resistant (MDR) Acinetobacter baumannii strains is among the most common and deadly forms of healthcare acquired infections. Over the years, different strategies have been put in place to increase host susceptibility to MDR A. baumannii, since only a self-limiting pneumonia with no or limited local bacterial replication was frequently obtained in mouse models. Direct instillation into the trachea or intranasal inoculation of the bacterial suspension are the techniques used to induce the infection in most of the preclinical models of pneumonia developed to date. More recently, the oropharyngeal aspiration procedure has been widely described in the literature for a variety of purposes including pathogens administration. Aim of this study was to compare the oropharyngeal aspiration technique to the intranasal inoculation and intratracheal instillation in the ability of inducing a consistent lung infection with two MDR A. baumannii clinical isolates in immunocompromised mice. Moreover, pneumonia obtained by bacteria administration with two out of three techniques, intratracheal and oropharyngeal, was characterised in terms of histopathology of pulmonary lesions, biomarkers of inflammation level and leukocytes cells infiltration extent after mice treatment with either vehicle or the antibiotic tigecycline. The data generated clearly showed that both strains were not able to colonize the lungs when inoculated by intranasal route. By contrast, the bacterial load in lungs of mice intratracheally or oropharyngeally infected significantly increased during 26 hours of monitoring, thus highlighting the ability of these strains to generate the infection when directly instilled into the lower respiratory airways. Furthermore, the intragroup variability of mice was significantly reduced with respect to those intranasally administered. Tigecycline was efficacious in lung bacterial load and cytokines release reduction. Findings were supported by semi-quantitative histopathological evaluation of the pulmonary lesions and by inflammatory biomarkers analysis. To conclude, both intratracheal instillation and oropharyngeal aspiration techniques showed to be suitable methods for inducing a robust and consistent pneumonia infection in mice when difficult MDR A. baumannii clinical isolates were used. Noteworthy, oropharyngeal aspiration not requiring specific technical skills and dedicated equipment, was proven to be a safer, easier and faster technique in comparison to the intratracheal instillation.

In Vitro Activity of Rifabutin and Rifampin against Antibiotic-Resistant Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

We recently reported that the antimicrobial activity of rifabutin against Acinetobacter baumannii is best modeled by the use of RPMI for in vitro susceptibility testing. Here, we define the effects of medium on the susceptibility and frequency of resistance emergence in a panel of A. baumannii, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa clinical isolates. Only A. baumannii was hypersusceptible to rifabutin in vitro and in vivo using a Galleria mellonella infection model. In vitro, the frequency of resistance emergence was greater when the bacteria were selected on RPMI versus tryptic soy agar (TSA) or Mueller-Hinton II (MHII) agar plates. However, the frequency of resistance emergence was lower in vivo than in the RPMI in vitro condition. IMPORTANCE Rifabutin has been recently described as a potential adjunctive therapy for antibiotic-resistant A. baumannii infections due to hypersensitivity in iron-depleted media, which may more closely mimic an in vivo environment. Here, we report that this hyperactivity is specific for A. baumannii, rather than being a general effect for other pathogens.

Controlling an Outbreak of Multidrug-resistant Acinetobacter baumannii in a Pediatric Intensive Care Unit: a Retrospective Analysis.

BACKGROUND: Multidrug-resistant Acinetobacter baumannii (MDRAB) is widespread among intensive care units worldwide, posing a threat to patients and the health system. We describe the successful management of a MDRAB outbreak by implementing an infection-control strategy in a pediatric intensive care unit (PICU). METHODS: This retrospective study investigated the patients admitted to the PICU in periods 1 (8 months) and 2 (7 months), from the index MDRAB case to intervention implementation, and from intervention implementation to cessation of MDRAB spread. An infection-control strategy was designed following six concepts: 1) cohort isolation of colonized patients, 2) enforcement of hand hygiene, 3) universal contact precautions, 4) environmental management, 5) periodic surveillance culture study, and 6) monitoring and feedback. RESULTS: Of the 427 patients, 29 were confirmed to have MDRAB colonization, of which 18 had MDRAB infections. Overall incidence per 1,000 patient days decreased from 7.8 (period 1) to 5.8 (period 2). The MDRAB outbreak was declared terminated after the 6-month follow-up following period 2. MDRAB was detected on the computer keyboard and in condensed water inside the ventilator circuits. The rate of hand hygiene performance was the lowest in the three months before and after index case admission and increased from 84% (period 1) to 95% (period 2). Patients with higher severity, indicated by a higher Pediatric Risk of Mortality III score, were more likely to develop colonization (P = 0.030), because they had invasive devices and required more contact with healthcare workers. MDRAB colonization contributed to an increase in the duration of mechanical ventilation and PICU stay (P < 0.001), but did not affect mortality (P = 0.273). CONCLUSION: The MDRAB outbreak was successfully terminated by the implementation of a comprehensive infection-control strategy focused on the promotion of hand hygiene, universal contact precautions, and environmental management through multidisciplinary teamwork.

Antimicrobial resistance profiles and associated factors of Acinetobacter and Pseudomonas aeruginosa nosocomial infection among patients admitted at Dessie comprehensive specialized Hospital, North-East Ethiopia. A cross-sectional study.

INTRODUCTION: Hospital admitted patients are at increased risk of nosocomial infections (NIs) with multi-drug resistant (MDR) pathogens which are prevalent in the hospital environment. Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii) are common causes of NIs worldwide. The objective of this study is to determine antimicrobial resistance profiles and associated factors of Acinetobacter spp and P. aeruginosa NIs among hospitalized patients. METHODS: A cross-sectional study was conducted at Dessie comprehensive specialized hospital, North-East Ethiopia, from February 1 to April 30, 2020. A total of 254 patients who were suspected of the bloodstream, urinary tract, or surgical site nosocomial infections were enrolled consecutively. Socio-demographic and other variables of interest were collected using a structured questionnaire. Specimens were collected and processed following standard microbiological procedures. Antimicrobial susceptibility was determined using the Kirby-Bauer disk diffusion method following Clinical and Laboratory Standards Institute guidelines. Data were analyzed with SPSS version 23 and p-value < 0.05 was considered statistically significant. RESULTS: Overall, 13% of patients had nosocomial Acinetobacter spp and/or P. aeruginosa infections. The culture positivity rate was 16(6.3%) for Acinetobacter spp and 18(7.1%) for P. aeruginosa. Patients admitted in the surgical ward (Adjusted odds ratio (AOR):10.66;95% confidence interval (CI):1.22-93.23), pediatric ward (AOR:14.37;95%CI:1.4-148.5), intensive care unit (AOR:41.93;95%CI:4.7-374.7) and orthopedics (AOR:52.21;95%CI:7.5-365) were significantly at risk to develop NIs compared to patients admitted in the medical ward. Patients who took more than two antimicrobial types at admission were 94% (AOR:0.06; 95% CI:0.004-0.84) times more protected from NIs compared to those who did not take any antimicrobial. About 81% of Acinetobacter spp and 83% of P. aeruginosa isolates were MDR. Amikacin and meropenem showed promising activity against Acinetobacter spp and P. aeruginosa isolates. CONCLUSION: The high prevalence of MDR Acinetobacter spp and P. aeruginosa nosocomial isolates enforce treating of patients with NIs based on antimicrobial susceptibility testing results.

Deciphering Multidrug-Resistant Acinetobacter baumannii from a Pediatric Cancer Hospital in Egypt.

Infection by multidrug-resistant (MDR) Acinetobacter baumannii is one of the major causes of hospital-acquired infections worldwide. The ability of A. baumannii to survive in adverse conditions as well as its extensive antimicrobial resistance make it one of the most difficult to treat pathogens associated with high mortality rates. The aim of this study was to investigate MDR A. baumannii that has spread among pediatric cancer patients in the Children's Cancer Hospital Egypt 57357. Whole-genome sequencing was used to characterize 31 MDR A. baumannii clinical isolates. Phenotypically, the isolates were MDR, with four isolates showing resistance to the last-resort antibiotic colistin. Multilocus sequence typing showed the presence of eight clonal groups, two of which were previously reported to cause outbreaks in Egypt, and one novel sequence type (ST), Oxf-ST2246. Identification of the circulating plasmids showed the presence of two plasmid lineages in the isolates, strongly governed by sequence type. A large number of antimicrobial genes with a range of resistance mechanisms were detected in the isolates, including ß-lactamases and antibiotic efflux pumps. Analysis of insertion sequences (ISs) revealed the presence of ISAba1 and ISAba125 in all the samples, which amplify ß-lactamase expression, causing extensive carbapenem resistance. Mutation analysis was used to decipher underlying mutations responsible for colistin resistance and revealed novel mutations in several outer membrane proteins, in addition to previously reported mutations in pmrB. Altogether, understanding the transmissibility of A. baumannii as well as its resistance and virulence mechanisms will help develop novel treatment options for better management of hospital-acquired infections. IMPORTANCE Acinetobacter baumannii represents a major health threat, in particular among immunocompromised cancer patients. The rise in carbapenem-resistant A. baumannii, and the development of resistance to the last-resort antimicrobial agent colistin, complicates the management of A. baumannii outbreaks and increases mortality rates. Here, we investigate 31 multidrug resistant A. baumannii isolates from pediatric cancer patients in Children's Cancer Hospital Egypt (CCHE) 57357 via whole-genome sequencing. Multilocus sequence typing (MLST) showed the presence of eight clonal groups including a novel sequence type. In silico detection of antimicrobial-resistant genes and virulence factors revealed a strong correlation between certain virulence genes and mortality as well as several point mutations in outer membrane proteins contributing to colistin resistance. Detection of CRISPR/Cas sequences in the majority of the samples was strongly correlated with the presence of prophage sequences and associated with failure of bacteriophage therapy. Altogether, understanding the genetic makeup of circulating A. baumannii is essential for better management of outbreaks.

Impact of target site mutations and plasmid associated resistance genes acquisition on resistance of Acinetobacter baumannii to fluoroquinolones.

Among bacterial species implicated in hospital-acquired infections are the emerging Pan-Drug Resistant (PDR) and Extensively Drug-Resistant (XDR) Acinetobacter (A.) baumannii strains as they are difficult to eradicate. From 1600 clinical specimens, only 100 A. baumannii isolates could be recovered. A high prevalence of ≥ 78% resistant isolates was recorded for the recovered isolates against a total of 19 tested antimicrobial agents. These isolates could be divided into 12 profiles according to the number of antimicrobial agents to which they were resistant. The isolates were assorted as XDR (68; 68%), Multi-Drug Resistant (MDR: 30; 30%), and PDR (2; 2%). Genotypically, the isolates showed three major clusters with similarities ranging from 10.5 to 97.8% as revealed by ERIC-PCR technique. As a resistance mechanism to fluoroquinolones (FQs), target site mutation analyses in gyrA and parC genes amplified from twelve selected A. baumannii isolates and subjected to sequencing showed 12 profiles. The selected isolates included two CIP-susceptible ones, these showed the wild-type profile of being have no mutations. For the ten selected CIP-resistant isolates, 9 of them (9/10; 90%) had 1 gyrA/1 parC mutations (Ser 81 → Leu mutation for gyrA gene and Ser 84 → Leu mutation for parC gene). The remaining CIP-resistant isolate (1/10; 10%) had 0 gyrA/1 parC mutation (Ser 84 → Leu mutation for parC gene). Detection of plasmid-associated resistance genes revealed that the 86 ciprofloxacin-resistant isolates carry qnrA (66.27%; 57/86), qnrS (70.93%; 61/86), aac (6')-Ib-cr (52.32%; 45/86), oqxA (73.25%; 63/86) and oqxB (39.53%; 34/86), while qepA and qnrB were undetected in these isolates. Different isolates were selected from profiles 1, 2, and 3 and qnrS, acc(6,)-ib-cr, oqxA, and oqxB genes harbored by these isolates were amplified and sequenced. The BLAST results revealed that the oqxA and oqxB sequences were not identified previously in A. baumannii but they were identified in Klebsiella aerogenes strain NCTC9793 and Klebsiella pneumoniae, respectively. On the other hand, the sequence of qnrS, and acc(6,)-ib-cr showed homology to those of A. baumannii. MDR, XDR, and PDR A. baumannii isolates are becoming prevalent in certain hospitals. Chromosomal mutations in the sequences of GyrA and ParC encoding genes and acquisition of PAFQR encoding genes (up to five genes per isolate) are demonstrated to be resistance mechanisms exhibited by fluoroquinolones resistant A. baumannii isolates. It is advisable to monitor the antimicrobial resistance profiles of pathogens causing nosocomial infections and properly apply and update antibiotic stewardship in hospitals and outpatients to control infectious diseases and prevent development of the microbial resistance to antimicrobial agents.

The Distribution of Multidrug-resistant Microorganisms and Treatment Status of Hospital-acquired Pneumonia/Ventilator-associated Pneumonia in Adult Intensive Care Units: a Prospective Cohort Observational Study.

BACKGROUND: It is essential to determine the distribution of the causative microorganisms in the region and the status of local antibiotic resistance for the proper treatment of hospital-acquired pneumonia/ventilator-associated pneumonia (HAP/VAP). This study aimed to investigate the occurrence and causative strains of HAP/VAP, distribution of resistant bacteria, use of antibiotics, and the ensuing outcomes of patients in Korea. METHODS: A multicenter prospective observational cohort study was conducted among patients with HAP/VAP admitted to the medical intensive care unit of 5 tertiary referral centers between August 2012 and June 2015. Patients' demographic and clinical data were collected. RESULTS: A total of 381 patients were diagnosed with HAP/VAP. Their median age was 69 (59-76) years and 71% were males. A majority of the patients (88%) had late-onset (> 5 days) HAP/VAP. One-quarter of the patients (n = 99) had at least one risk factor for multidrug-resistant (MDR) pathogens, such as prior intravenous antibiotic use within the last 90 days. Microbiological specimens were mostly obtained noninvasively (87%) using sputum or endotracheal aspirates. Pathogens were identified in 235 (62%) of the 381 patients. The most common bacterial pathogen was Acinetobacter baumannii (n = 89), followed by Staphylococcus aureus (n = 52), Klebsiella pneumoniae (n = 25) and Pseudomonas aeruginosa (n = 22). Most of isolated A. baumannii (97%) and S. aureus (88%) were multidrug resistant. The most commonly used empirical antibiotic regimens were carbapenem-based antibiotics (38%), followed by extended-spectrum penicillin/ß-lactamase inhibitor (34%). Glycopeptide or linezolid were also used in combination in 54% of patients. The 28-day mortality rate of the patients with HAP/VAP was 30% and the 60-day mortality was 46%. Patients who used empirical antibiotics appropriately had significantly lower mortality rates than those who did not (28-day mortality: 25% vs. 40%, P = 0.032; 60-day mortality: 41% vs. 55%, P = 0.032, respectively). Administration of appropriate empirical antibiotics (odds ratio [OR], 0.282; confidence interval [CI], 0.092-0.859; P = 0.026), Day 7 treatment failure (OR, 4.515; CI, 1.545-13.192; P = 0.006), and APACHE II score on day 1 (OR, 1.326; CI, 0.988-1.779; P = 0.012) were the factors that determined the 28-day mortality in patients with HAP who had identified bacteria as pathogens. CONCLUSION: In HAP/VAP patients, there was a large burden of MDR pathogens, and their associated mortality rate was high. Proper selection of empirical antibiotics was significantly associated with the patient's prognosis; however, there was a discrepancy between major pathogens and empirical antibiotic therapy.

Tigecycline Heteroresistance and Resistance Mechanism in Clinical Isolates of Acinetobacter baumannii.

Tigecycline is regarded as a last-resort treatment for multidrug-resistant Acinetobacter baumannii. However, tigecycline resistance in A. baumannii has increased worldwide. In this study, we investigated tigecycline heteroresistance in A. baumannii isolates from South Korea. Antibiotic susceptibility testing was performed on 323 nonduplicated A. baumannii isolates. Among 260 and 37 tigecycline-susceptible and -intermediate-resistant A. baumannii isolates, 146 (56.2%) and 22 (59.5%) isolates were identified as heteroresistant to tigecycline through a disk diffusion assay and population analysis profiling. For selected isolates, an in vitro time-kill assay was performed, and survival rates were measured after preincubation with diverse concentrations of tigecycline. Heteroresistant isolates showed regrowth after 12 h of 2× MIC of tigecycline treatment, and resistant subpopulations were selected by preexposure to tigecycline. Furthermore, genetic alterations in adeABC, adeRS, and rpsJ were assessed, and the relative mRNA expression levels of adeB and adeS were compared. The tigecycline resistance in subpopulations might be due to the insertion of ISAba1 in adeS, leading to the overexpression of the AdeABC efflux pump. However, the tigecycline resistance of subpopulations was not stable during serial passages in antibiotic-free medium. The reversion of tigecycline susceptibility by antibiotic-free passages might occur by additional insertions of ISAba10 in adeR and nucleotide alterations in adeS in some mutants. Tigecycline heteroresistance is prevalent in A. baumannii isolates, which results in treatment failure. Tigecycline resistance is mainly due to the overexpression of the AdeABC efflux pump, which is associated with genetic mutations, but this resistance could be reversed into susceptibility by additional mutations in antibiotic-free environments. IMPORTANCE The evidence that antibiotic heteroresistance is responsible for treatment failure in clinical settings is increasing. Thus, detection and characterization of heteroresistance would be important for appropriate therapeutic guidance to treat bacterial infections. However, data on tigecycline heteroresistance in Gram-negative bacteria is currently limited, although tigecycline is regarded as a last-line antibiotic against infections caused by antibiotic-resistant pathogens. In this study, we investigated the tigecycline heteroresistance in Acinetobacter baumannii, which has been listed by the WHO as a priority for research and development of new antibiotics. We found very high prevalence of tigecycline-heteroresistant A. baumannii clinical isolates, which may result in treatment failure due to the selection of resistant subpopulations. We also identified the main resistance mechanism in tigecycline-resistant subpopulations, that is, upregulation of AdeABC efflux pumps due to ISAba1 insertion in adeS.

Antibacterial effect of acetic acid during an outbreak of carbapenem-resistant Acinetobacter baumannii in an ICU (II).

INTRODUCTION: Acetic acid (AA) has been commonly used in medicine as an antiseptic agent for the past 6000 years. This study evaluated the antibacterial effect of AA during an outbreak in an intensive care unit (ICU) facility in Baja California Sur, México. METHODOLOGY: Thirty-five environmental samples were collected, subsequently, disinfection with AA (4%) was performed, and two days later the same areas were sampled inside the ICU facility. Carbapenem-resistant A. baumannii (CRAB) was detected with loop-mediated isothermal amplification assay (Garciglia-Mercado et al. companion paper), targeting blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP and blaVIM genes. CRAB isolates before and after disinfection were compared by PFGE. RESULTS: Eighteen (54.5%) and five (14.3%) of thirty-five environmental samples were identified as Acinetobacter baumannii before and after disinfection, respectively, showing a significant decrease of 85.7% (p < 0.05) both by Loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Furthermore, the presence of blaOXA-23-like and blaOXA-58-like genes significantly decreased (p < 0.05) both by LAMP and PCR methods. PFGE genotype showed high similarity among CRAB isolates before and after disinfection, suggesting wide clonal dissemination in the ICU facility. CONCLUSIONS: This study demonstrated the novel application of AA with the LAMP assays developed for detecting CRAB. AA promises to be a cheap and efficacious disinfectant alternative to both developed and especially developing countries, preventing the spread of this organism in the environment and to other susceptible patients in health care settings.

A LAMP-based system for rapid detection of eight common pathogens causing lower respiratory tract infections.

Lower respiratory tract infections (LRTIs) are a leading cause of morbidity and mortality worldwide and lack a rapid diagnostic method. To improve the diagnosis of LRTIs, we established an available loop-mediated isothermal amplification (LAMP) assay for the detection of eight common lower respiratory pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. The whole process can be achieved within 1 h (sample to results read out). We established an extraction free isothermal system. 528 sputum samples collected from patients suspected to have LRTIs were analyzed by the system (8 tests in each sample, a total of 4224 tests) and compared with the standard culture method (SCM). The samples with inconsistent results were further verified by Sanger sequencing and High-throughput sequencing (NGS). The detection limits of the LAMP assay for the 8 pathogens ranged from 103 to 104 CFU/mL. Upon testing 528 samples, the Kappa coefficients of all pathogens ranged between 0.5 and 0.7 indicated a moderate agreement between the LAMP assay and the SCM. All inconsistent samples were further verified by Sanger sequencing, we found that the developed LAMP assay had a higher consistency level with Sanger sequencing than the SCM for all pathogens. Additionally, when the NGS was set to a diagnostic gold standard, the specificity and sensitivity of the LAMP assay for LRTIs were 94.49% and 75.00%. The present study demonstrated that the developed LAMP has high consistency with the sequencing methods. Meanwhile, the LAMP assay has a higher detection rate compared to the SCM. It may be a powerful tool for rapid and reliable clinical diagnosis of LRTIs in primary hospitals.

The investigation of adrenal involvement in carbapenem resistant Acinetobacter baumannii sepsis: experimental mouse model

Background/aim: In the last years, incidence of carbapenem resistant Acinetobacter baumannii sepsis is increasing with high mortality. However, it is not clear whether this is due to inadequate antimicrobial choice or a more severe clinical course. We aimed to evaluate the inflammation and adrenal involvement in the carbapenem resistant A. baumannii by using experimental mouse model sepsis. Materials and methods: Balb/c female mice were randomly put into control and three sepsis groups ( A. baumannii susceptible to carbapenem-CSAB-, A. baumannii resistant to carbapenem-CRAB-, Escherichia coli). A total of sixty mice were included in this study with each group having 15 mice. Mice were sacrificed 72 h after bacterial inoculation, and blood was taken from each mouse for the assessment of cytokines and corticosterone. Both adrenal glands were dissected; one was used for culture and the other was used for histopathological examination. Bacterial loads of organs were calculated as CFU/g. The histopathological changes, bacterial levels in adrenal and cytokine and corticosterone levels were assessed and compared among the groups. Results: The bacterial level was higher in E. coli (108, 45 ±30, 55 log10 CFU/g) (mean±SD) than other sepsis groups. The lowest level of corticosterone was observed in the E. coli group (p < 0.001). TNF alpha level was highest in the CRAB and E. coli group and this difference was statistically significant than control group (p < 0.05). The IL-6 level in CRAB was significantly higher than the control group (10, 20 pg/mL). The adrenal gland congestion was significantly severe in all the sepsis groups compared to the control. In the group comparison, congestion was significantly more severe in the E. coli group than in CSAB and CRAB groups. Conclusion: Adrenal involvement and inflammatory reactions are seen in E. coli sepsis and in CRAB sepsis. These findings will be focused on in future clinical trials.