A bioinformatic approach to identify confirmed and probable CRISPR-Cas systems in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex genomes.

Introduction: The Acinetobacter calcoaceticus-Acinetobacter baumannii complex, or Acb complex, consists of six species: Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii, and Acinetobacter lactucae. A. baumannii is the most clinically significant of these species and is frequently related to healthcare-associated infections (HCAIs). Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and associated genes (cas) constitute bacterial adaptive immune systems and function as variable genetic elements. This study aimed to conduct a genomic analysis of Acb complex genomes available in databases to describe and characterize CRISPR systems and cas genes. Methods: Acb complex genomes available in the NCBI and BV-BRC databases, the identification and characterization of CRISPR-Cas systems were performed using CRISPRCasFinder, CRISPRminer, and CRISPRDetect. Sequence types (STs) were determined using the Oxford scheme and ribosomal multilocus sequence typing (rMLST). Prophages were identified using PHASTER and Prophage Hunter. Results: A total of 293 genomes representing six Acb species exhibited CRISPR-related sequences. These genomes originate from various sources, including clinical specimens, animals, medical devices, and environmental samples. Sequence typing identified 145 ribosomal multilocus sequence types (rSTs). CRISPR-Cas systems were confirmed in 26.3% of the genomes, classified as subtypes I-Fa, I-Fb and I-Fv. Probable CRISPR arrays and cas genes associated with CRISPR-Cas subtypes III-A, I-B, and III-B were also detected. Some of the CRISPR-Cas systems are associated with genomic regions related to Cap4 proteins, and toxin-antitoxin systems. Moreover, prophage sequences were prevalent in 68.9% of the genomes. Analysis revealed a connection between these prophages and CRISPR-Cas systems, indicating an ongoing arms race between the bacteria and their bacteriophages. Furthermore, proteins associated with anti-CRISPR systems, such as AcrF11 and AcrF7, were identified in the A. baumannii and A. pittii genomes. Discussion: This study elucidates CRISPR-Cas systems and defense mechanisms within the Acb complex, highlighting their diverse distribution and interactions with prophages and other genetic elements. This study also provides valuable insights into the evolution and adaptation of these microorganisms in various environments and clinical settings.

Molecular Epidemiology of Acinetobacter calcoaceticus-Acinetobacter baumannii Complex Isolated From Children at the Hospital Infantil de México Federico Gómez.

The Acinetobacter calcoaceticus-baumannii (Acb) complex is regarded as a group of phenotypically indistinguishable opportunistic pathogens responsible for mainly causing hospital-acquired pneumonia and bacteremia. The aim of this study was to determine the frequency of isolation of the species that constitute the Acb complex, as well as their susceptibility to antibiotics, and their distribution at the Hospital Infantil de Mexico Federico Gomez (HIMFG). A total of 88 strains previously identified by Vitek 2®, 40 as Acinetobacter baumannii and 48 as Acb complex were isolated from 52 children from 07, January 2015 to 28, September 2017. A. baumannii accounted for 89.77% (79/88) of the strains; Acinetobacter pittii, 6.82% (6/88); and Acinetobacter nosocomialis, 3.40% (3/88). Most strains were recovered mainly from patients in the intensive care unit (ICU) and emergency wards. Blood cultures (BC) provided 44.32% (39/88) of strains. The 13.63% (12/88) of strains were associated with primary bacteremia, 3.4% (3/88) with secondary bacteremia, and 2.3% (2/88) with pneumonia. In addition, 44.32% (39/88) were multidrug-resistant (MDR) strains and, 11.36% (10/88) were extensively drug-resistant (XDR). All strains amplified the bla OXA-51 gene; 51.13% (45/88), the bla OXA-23 gene; 4.54% (4/88), the bla OXA-24 gene; and 2.27% (2/88), the bla OXA-58 gene. Plasmid profiles showed that the strains had 1-6 plasmids. The strains were distributed in 52 pulsotypes, and 24 showed identical restriction patterns, with a correlation coefficient of 1.0. Notably, some strains with the same pulsotype were isolated from different patients, wards, or years, suggesting the persistence of more than one clone. Twenty-seven sequence types (STs) were determined for the strains based on a Pasteur multilocus sequence typing (MLST) scheme using massive sequencing; the most prevalent was ST 156 (27.27%, 24/88). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas I-Fb system provided amplification in A. baumannii and A. pittii strains (22.73%, 20/88). This study identified an increased number of MDR strains and the relationship among strains through molecular typing. The data suggest that more than one strain could be causing an infection in some patient. The implementation of molecular epidemiology allowed the characterization of a set of strains and identification of different attributes associated with its distribution in a specific environment.

Long-Term Surveillance of Antibiotic Prescriptions and the Prevalence of Antimicrobial Resistance in Non-Fermenting Gram-Negative Bacilli.

The increasing emergence of multidrug-resistant (MDR) bacteria has been recognized as a public health threat worldwide. Hospitalized patients and outpatients are commonly infected by non-fermenting Gram-negative bacilli (NFGNB), particularly the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ACB) and Pseudomonas aeruginosa. Antimicrobial agents are critical for treating the nosocomial infections caused by NFGNB. The aim of this study was to assess antimicrobial resistance and the use of antimicrobial agents. The bacterial isolates of 638,152 specimens from both inpatients and outpatients, retrieved from 2001 to 2008 at a medical center in central Taiwan, were examined for their susceptibility to various antimicrobial agents, including cefepime, imipenem, ciprofloxacin, gentamicin, amikacin, meropenem, and levofloxacin. Administrated prescriptions of the monitored antibiotics were analyzed using the Taiwan National Health Insurance Research Database (NHIRD). Our results show that the defined daily doses (DDDs) for cefepime, imipenem, and ciprofloxacin increased with time, and a trend toward reduced antimicrobial sensitivities of both ACB and P. aeruginosa was noticeable. In conclusion, the antimicrobial sensitivities of ACB and P. aeruginosa were reduced with the increased use of antibiotics. Continuous surveillance of antibiotic prescriptions and the prevalence of emerging resistance in nosocomial infections is warranted.

Genetic Characterization of Plasmid-Borne blaOXA-58 in Distinct Acinetobacter Species.

We characterize by whole-plasmid-sequence (WPS) two-plasmid-borne blaOXA-58 obtained from Acinetobacter seifertii (Asp-1069) and A. baumannii (Acb-45063) clinical strains recovered 17 years apart from distinct Brazilian regions. Multilocus sequence type (MLST) analysis showed that the Asp-1069 and Acb-45063 strains belong to ST551 and ST15/CC15, respectively. WPS analysis demonstrated that blaOXA-58 was located in two distinct plasmids named pAs1069_a (24,672 bp/44 open reading frames [ORFs]) and pAb45063_b (19,808 bp/24 ORFs), which belong to the GR8/GR23 (repAci23) and GR4 (repAci4) incompatibility groups, respectively. The genetic environments surrounding blaOXA-58 revealed that it was flanked by two intact ISAba3 copies on pAb45063_b, which differed from pAs1069_a. In the latter, the upstream ISAba3 copy was truncated by insertion of ISAba825 element. Although Re27-specific recombination sites were found adjacent to ISAba3-blaOXA-58-ISAba3 arrangement on pAb45063_b, such structures were absent on pAs1069_a. The conserved ISAba125-araC1-lysE arrangement was disrupted by TnaphA6 harboring the aminoglycosides resistance gene aphA6 on pAs1069_a, while an IS26-blaTEM-1-aac(3)-IIa-IS26 genetic structure was found upstream from ISAba3-blaOXA-58-ISAba3 on pAb45063_b. Other two plasmids, pAb45063_a (183,767 bp/209 ORFs) and pAs1069_b (13,129 bp/14 ORFs), were also found in the OXA-58-producing Acinetobacter species strains, harboring the strA and strB genes and the sul2 gene, which confer resistance to streptomycin and sulfonamides, respectively. The plasmid-mediated virulence factors corresponding to genes tonB, spl, glmM, ppa, sulP, and map were found in both strains, as well distinct toxin-antitoxin system-encoding genes stbD and relE (pAs1069_a), brnT and brnA (pAb45063_b), and xreE (pAb45063_a). Although infrequently reported in Brazil, plasmid-borne blaOXA-58 showed a complex and diverse genetic backbone that confers stability in different Acinetobacter species that have been isolated from nosocomial settings over time.IMPORTANCE Although the blaOXA-58 gene has been infrequently described in Brazil, contrasting with other bordering South American countries, we verified the maintenance of this resistance determinant over time among carbapenem-resistant Acinetobacter species isolates, not only in nosocomial settings but also in the environment. In addition, to the best of our knowledge, this is the first study to have used WPS analysis to evaluate the genetic surroundings of blaOXA-58 in Brazil. Moreover, the A. seifertii and A. baumannii clinical strains evaluated in this study were recovered 17 years apart in hospitals located in distinct Brazilian geographic regions.

Acinetobacter pittii isolated more frequently than Acinetobacter baumannii in blood cultures: the experience of a French hospital.

This study assessed the characteristics of hospital-acquired Acinetobacter calcoaceticus-Acinetobacter baumannii complex bloodstream infections (BSI). From 2010 to 2017, A. calcoaceticus-A. baumannii complex BSI were investigated. Of 73 cases, 54.8% were associated with Acinetobacter pittii, 39.7% with Acinetobacter baumannii and 5.5% with Acinetobacter nosocomialis. Multi-drug-resistant isolates were significantly higher in A. baumannii BSI. Thirty-day mortality was no different between A. baumannii or non-baumannii A. calcoaceticus-A. baumannii complex BSI. In contrast with other studies, this study found that most hospital-acquired A. calcoaceticus-A. baumannii complex BSI were associated with non-baumannii A. calcoaceticus-A. baumannii complex species. This study found that these species were important hospital-acquired pathogens, and emphasizes the importance of A. calcoaceticus-A. baumannii complex species identification.

The role of the genetic elements bla oxa and IS Aba 1 in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex in carbapenem resistance in the hospital setting

Abstract: INTRODUCTION: Members of the Acinetobacter genus are key pathogens that cause healthcare-associated infections, and they tend to spread and develop new antibiotic resistance mechanisms. Oxacillinases are primarily responsible for resistance to carbapenem antibiotics. Higher rates of carbapenem hydrolysis might be ascribed to insertion sequences, such as the ISAba1 sequence, near bla OXA genes. The present study examined the occurrence of the genetic elements bla OXA and ISAba1 and their relationship with susceptibility to carbapenems in clinical isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. METHODS: Isolates identified over 6 consecutive years in a general hospital in Joinville, Southern Brazil, were evaluated. The investigation of 5 families of genes encoding oxacillinases and the ISAba1 sequence location relative to bla OXA genes was conducted using polymerase chain reaction. RESULTS: All isolates presented the bla OXA-51-like gene (n = 78), and 91% tested positive for the bla OXA-23-like gene (n = 71). The presence of ISAba1 was exclusively detected in isolates carrying the bla OXA-23-like gene. All isolates in which ISAba1 was found upstream of the bla OXA-23-like gene (n = 69) showed resistance to carbapenems, whereas the only isolate in which ISAba1 was not located near the bla OXA-23-like gene was susceptible to carbapenems. The ISAba1 sequence position of another bla OXA-23-like-positive isolate was inconclusive. The isolates exclusively carrying the bla OXA-51-like gene (n = 7) showed susceptibility to carbapenems. CONCLUSIONS: The presence of the ISAba1 sequence upstream of the bla OXA-23-like gene was strongly associated with carbapenem resistance in isolates of the A. calcoaceticus-A. baumannii complex in the hospital center studied.

Sulbactam treatment for pneumonia involving multidrug-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

BACKGROUND: Multidrug-resistant (MDR) Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex has become an important cause of nosocomial pneumonia. Sulbactam is a ß-lactamase inhibitor with antimicrobial activity against MDR Acb complex. METHODS: To investigate outcomes of pneumonia involving MDR Acb complex treated with sulbactam or ampicillin/sulbactam for at least 7 days, we conducted a retrospective study of 173 adult patients over a 34 month period. RESULTS: Of 173 patients, 138 (79.8%) received combination therapy, mainly with carbapenems (119/138, 86.2%). The clinical response rate was 67.6% and the 30 day mortality rate was 31.2%. The independent predictors of clinical failure were malignancy, bilateral pneumonia and shorter duration of treatment. In patients with sulbactam-susceptible strains, there was no difference in clinical and microbiological outcome between combination therapy and monotherapy. Compared to the sulbactam-susceptible group, the sulbactam-resistant group had a lower rate of airway eradication, a longer duration of treatment and a higher rate of combination therapy with predominantly carbapenems (p < 0.05). There was no significant difference between the two groups in clinical resolution and 30 day mortality rates. CONCLUSIONS: Sulbactam could be a treatment option for pneumonia involving MDR Acb complex, and combination therapy with carbapenems could be considered for sulbactam-resistant cases.

Improvement of MALDI-TOF MS profiling for the differentiation of species within the Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

MALDI-TOF MS is currently becoming the method of choice for rapid identification of bacterial species in routine diagnostics. Yet, this method suffers from the inability to differentiate reliably between some closely related bacterial species including those of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, namely A. baumannii and Acinetobacter nosocomialis. In the present study, we evaluated a protocol which was different from that used in the Bruker Daltonics identification system (MALDI BioTyper) to improve species identification using a taxonomically precisely defined set of 105 strains representing the four validly named species of the ACB complex. The novel protocol is based on the change in matrix composition from alpha-cyano-4-hydroxycinnamic acid (saturated solution in water:acetonitrile:trifluoroacetic acid, 47.5:50:2.5, v/v) to ferulic acid (12.5mgml(-1) solution in water:acetonitrile:formic acid 50:33:17, v/v), while the other steps of sample processing remain unchanged. Compared to the standard protocol, the novel one extended the range of detected compounds towards higher molecular weight, produced signals with better mass resolution, and allowed the detection of species-specific signals. As a result, differentiation of A. nosocomialis and A. baumannii strains by cluster analysis was improved and 13 A. nosocomialis strains, assigned erroneously or ambiguously by using the standard protocol, were correctly identified.

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.