The mechanism of survival and degradation of phenol by Acinetobacter pittii in an extremely acidic environment.

The treatment efficiency of acidic phenol-containing wastewater is hindered by the absence of efficient acid-resistant phenol-degrading bacteria, and the acid-resistant mechanism of such bacteria remains poorly studied. In this study, the acid-resistant strain Hly3 was used as a research model to investigate its ability to degrade phenol and its underlying mechanism of acid resistance. Strain Hly3 exhibited robust acid resistance, capable of surviving in extremely acidic environments (pH 3) and degrading 1700 mg L-1 phenol in 72 h. Through the physiological response analysis of strain Hly3 to pH, the results indicated: firstly, the strain could reduce the relative permeability of the cell membrane and increase EPS secretion to prevent H+ from entering the cell (shielding effect); secondly, the strain could accumulate more buffering substances to neutralize the intracellular H+ (neutralization effect); thirdly, the strain could expel H+ from the cell by enhancing H+-ATPase activity (pumping effect); finally, the strain produced more active scavengers to reduce the toxicity of acid stress on cells (antioxidant effect). Subsequently, combining liquid chromatography-mass spectrometry (LC-MS) technology with exogenous addition experiments, it was verified that the acid resistance mechanism of microorganisms is achieved through the activation of acid-resistant response systems by glutamine, thereby enhancing functions such as shielding, neutralization, efflux, and antioxidation. This study elucidated the acid resistance mechanism of Acinetobacter pittii, providing a theoretical basis and guidance for the treatment of acidic phenol-containing wastewater.

Characterization of Diesel Degrading Indigenous Bacterial Strains, Acinetobacter pittii and Pseudomonas aeruginosa, Isolated from Oil Contaminated Soils.

In this study, 13 diesel degrading bacteria were isolated from the oil contaminated soils and the promising strains identified as Acinetobacter pittii ED1 and Pseudomonas aeruginosa BN were evaluated for their diesel degrading capabilities. These strains degraded the diesel optimally at 30 °C, pH 7.0 and 1% diesel concentration. Both the strains produced biofilm at 1% diesel concentration indicating their ability to tolerate diesel induced abiotic stress. Gravimetric analysis of the spent medium after 7 days of incubation showed that A. pittii ED1 and P. aeruginosa BN degraded 68.61% and 76% diesel, respectively, while biodegradation reached more than 90% after 21 days. Fourier Transform Infrared (FTIR) analysis of the degraded diesel showed 1636.67 cm-1 (C=C stretch, N-H bond) peak corresponding to alkenes and primary amines, while GC-TOF-MS analysis showed decline in hydrocarbon intensities after 7 days of incubation. The present study revealed that newly isolated A. pittii ED1 and P. aeruginosa BN were able to degrade diesel hydrocarbons (C11-C18, and C19-C24) efficiently and have potential for bioremediation of the oil-contaminated sites. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01317-3.

Genetic characterization of plasmid-borne blaOXA-58 and blaOXA-72 in Acinetobacter pittii in Shaanxi, China.

OBJECTIVES: Acinetobacter pittii has emerged as an opportunistic nosocomial pathogen associated with hospital-acquired infections. The purpose of this study was to investigate the genetic structures of plasmids carrying carbapenemase genes blaOXA-58 and blaOXA-72 in A. pittii strains AR3676 and AR3651 isolated from patients. METHODS: Antimicrobial susceptibility testing was performed using broth microdilution. Whole-genome sequencing and bioinformatics analysis were performed to characterize the genome of A. pittii AR3676 and AR3651. Conjugation experiments were conducted to evaluate plasmid transferability. Phylogenetic and comparative genomic analysis were performed to explore the characteristics of carbapenem-resistant A. pittii isolates worldwide. RESULTS: The AR3676 strain showed resistance to imipenem. The 19 700-bp plasmid pAR3676-OXA-58 harboured blaOXA-58 with genetic contexts consisting of a truncated ISAba3-like-blaOXA58-ISAba3. Additionally, the AR3651 strain showed resistance to imipenem and meropenem. The AR3651 genome comprised one 9,837-bp RepA_AB plasmid pAR3651-OXA-72 harbouring blaOXA-72. This blaOXA-72 was flanked by XerC/XerD recombination sites. The conjugation of plasmids pAR3676-OXA-58 and pAR3651-OXA-72 from A. pittii to Acinetobacter baumannii ATCC 17978RIFR failed three independent times. Phylogenetic analysis of A. pittii strains AR3676, AR3651, and a further 504 A. pittii strains collected between 1966 and 2022 from various geographic localities revealed genetic diversity with a heterogeneous distribution of carbapenemase genes. CONCLUSIONS: A. pittii strains with a plasmid carrying blaOXA-58 or blaOXA-72 may serve as an important reservoir of carbapenemase genes. Carbapenemase genes on a single plasmid may facilitate their dissemination and persistence. Additionally, pdif sites and mobile elements play an important role in the mobilization of resistance genes and plasmid evolution.

Identification and characterization of the capsule depolymerase Dpo27 from phage IME-Ap7 specific to Acinetobacter pittii.

Among the Acinetobacter genus, Acinetobacter pittii stands out as an important opportunistic infection causative agent commonly found in hospital settings, which poses a serious threat to human health. Recently, the high prevalence of carbapenem-resistant A. pittii isolates has created significant therapeutic challenges for clinicians. Bacteriophages and their derived enzymes are promising therapeutic alternatives or adjuncts to antibiotics effective against multidrug-resistant bacterial infections. However, studies investigating the depolymerases specific to A. pittii strains are scarce. In this study, we identified and characterized a capsule depolymerase, Dpo27, encoded by the bacteriophage IME-Ap7, which targets A. pittii. A total of 23 clinical isolates of Acinetobacter spp. were identified as A. pittii (21.91%, 23/105), and seven A. pittii strains with various K locus (KL) types (KL14, KL32, KL38, KL111, KL163, KL207, and KL220) were used as host bacteria for phage screening. The lytic phage IME-Ap7 was isolated using A. pittii 7 (KL220) as an indicator bacterium and was observed for depolymerase activity. A putative tail fiber gene encoding a polysaccharide-degrading enzyme (Dpo27) was identified and expressed. The results of the modified single-spot assay showed that both A. pittii 7 and 1492 were sensitive to Dpo27, which was assigned the KL220 type. After incubation with Dpo27, A. pittii strain was susceptible to killing by human serum; moreover, the protein displayed no hemolytic activity against erythrocytes. Furthermore, the protein exhibited sustained activity across a wide pH range (5.0-10.0) and at temperatures between 20 and 50°C. In summary, the identified capsule depolymerase Dpo27 holds promise as an alternative treatment for combating KL220-type A. pittii infections.

First report of Acinetobacter pittii acute community-acquired pneumonia in an immunocompetent patient in France following a heat wave.

BACKGROUND: In recent years, Acinetobacter baumannii-calcoaceticus complex (ABC) infections have attracted attention, mainly because of the impact of carbapenem-resistant isolates in hospital-acquired infections. However, acute community-acquired ABC infections are not uncommon in warm and humid countries, where they are responsible for community-acquired infections with specific clinical features. To date, such infection has not been reported in France. CASE PRESENTATION: We report the case of a 55-year-old non-immunocompromised patient living in France with no known risk factors for community-acquired ABC infections who presented pneumonia with bloodstream infection due to wild-type A. pittii. The outcome was favorable after 7 days of antibiotic treatment with cefepime. We confirmed bacterial identification with whole-genome sequencing, and we examined the A. pitii core-genome phylogeny for genomic clusters. CONCLUSIONS: This situation is uncommon in Europe and occurred after a heat wave in France with temperatures above 38 °C. Herein, we discuss the possibility that this pneumonia may be emerging in the current context of global warming.

Isolation, identification, degradation mechanism and exploration of active enzymes in the ochratoxin A degrading strain Acinetobacter pittii AP19.

Ochratoxin A (OTA) is a polyketide mycotoxin that commonly contaminates agricultural products and causes significant economic losses. In this study, the efficient OTA-degrading strain AP19 was isolated from vineyard soil and was identified as Acinetobacter pittii. Compared with growth in nutrient broth supplemented with OTA (OTA-NB), strain AP19 grew faster in nutrient broth (NB), but the ability of the resulting cell lysates to remove OTA was weaker. After cultivation in NB, the cell lysate of strain AP19 was able to remove 100% of 1 mg/L OTA within 18 h. The cell lysate fraction > 30 kDa degraded 100% of OTA within 12 h, while the fractions < 30 kDa were practically unable to degrade OTA. Further anion exchange chromatography of the > 30 kDa fraction yielded two peaks exhibiting significant OTA degradation activity. The degradation product was identified as OTα. Amino acid metabolism exhibited major transcriptional trends in the response of AP19 to OTA. The dacC gene encoding carboxypeptidase was identified as one of the contributors to OTA degradation. Soil samples inoculated with strain AP19 showed significant OTA degradation. These results provide significant insights into the discovery of novel functions in A. pittii, as well as its potential as an OTA decomposer.

Identification of an Acinetobacter pittii acyltransferase involved in transformation of deoxynivalenol to 3-acetyl-deoxynivalenol by transcriptomic analysis.

Deoxynivalenol (DON), a mycotoxin primarily produced by Fusarium graminearum (F. graminearum), is widely present in food and feed, posing great hazards to human and livestock health. In this study, a strain of Acinetobacter pittii (A. pittii) S12 capable of degrading DON was isolated from soil samples and identified through morphological characterization, biochemistry analysis, and 16 S rRNA gene sequencing. The results of HPLC-MS indicated that the degradation products underwent a conversion from [M-H]- to [M+CH3CO], with concomitant transformation of the hydroxyl group into an acetyl moiety. Based on transcriptome sequencing analysis, the acyltransferase encoded by DLK06_RS13370 was predicted to be the pivotal gene responsible for DON biotransformation. The result of molecular docking analysis suggest a high affinity between the enzyme and DON. The recombinant protein encoded by DLK06_RS13370 was expressed in Escherichia coli (E. coli) and demonstrated the capacity to catalyze the conversion of DON into 3-Acetyl-deoxynivalenol (3-ADON), as confirmed by HPLC analysis. In conclusion, our findings confirm that the acyltransferase encoded by DLK06-RS13370 is responsible for the acetylation of DON. This sheds light on the co-occurrence of DON and its acetyl-derivatives in wheat-based products. DATA AVAILABILITY: Not applicable.

Emergence of uncommon KL38-OCL6-ST220 carbapenem-resistant Acinetobacter pittii strain, co-producing chromosomal NDM-1 and OXA-820 carbapenemases.

Objective: To characterize one KL38-OCL6-ST220 carbapenem-resistant Acinetobacter pittii strain, co-producing chromosomal NDM-1 and OXA-820 carbapenemases. Methods: A. pittii TCM strain was isolated from a bloodstream infection (BSI). Antimicrobial susceptibility tests were conducted via disc diffusion and broth microdilution. Stability experiments of bla NDM-1 and bla OXA-820 carbapenemase genes were further performed. Whole-genome sequencing (WGS) was performed on the Illumina and Oxford Nanopore platforms. Multilocus sequence typing (MLST) was analyzed based on the Pasteur and Oxford schemes. Resistance genes, virulence factors, and insertion sequences (ISs) were identified with ABRicate based on ResFinder 4.0, virulence factor database (VFDB), and ISfinder. Capsular polysaccharide (KL), lipooligosaccharide outer core (OCL), and plasmid reconstruction were tested using Kaptive and PLACNETw. PHASTER was used to predict prophage regions. A comparative genomics analysis of all ST220 A. pittii strains from the public database was carried out. Point mutations, average nucleotide identity (ANI), DNA-DNA hybridization (DDH) distances, and pan-genome analysis were performed. Results: A. pittii TCM was ST220Pas and ST1818Oxf with KL38 and OCL6, respectively. It was resistant to imipenem, meropenem, and ciprofloxacin but still susceptible to amikacin, colistin, and tigecycline. WGS revealed that A. pittii TCM contained one circular chromosome and four plasmids. The Tn125 composite transposon, including bla NDM-1, was located in the chromosome with 3-bp target site duplications (TSDs). Many virulence factors and the bla OXA-820 carbapenemase gene were also identified. The stability assays revealed that bla NDM-1 and bla OXA-820 were stabilized by passage in an antibiotic-free medium. Moreover, 12 prophage regions were identified in the chromosome. Phylogenetic analysis showed that there are 11 ST220 A. pittii strains, and one collected from Anhui, China was closely related. All ST220 A. pittii strains presented high ANI and DDH values; they ranged from 99.85% to 100% for ANI and from 97.4% to 99.9% for DDH. Pan-genome analysis revealed 3,200 core genes, 0 soft core genes, 1,571 shell genes, and 933 cloud genes among the 11 ST220 A. pittii strains. Conclusions: The coexistence of chromosomal NDM-1 and OXA-820 carbapenemases in A. pittii presents a huge challenge in healthcare settings. Increased surveillance of this species in hospital and community settings is urgently needed.

Genomic analysis of Acinetobacter pittii CEP14 reveals its extensive biodegradation capabilities, including cometabolic degradation of cis-1,2-dichloroethene.

Halogenated organic compounds are naturally occurring in subsurface environments; however, accumulation of the degradative intermediate cis-1,2-dichloroethene (cDCE) at soil and groundwater sites contaminated with xenobiotic chlorinated ethenes is a global environmental and public health issue. Identifying microorganisms capable of cDCE degradation in these environments is of interest because of their potential application to bioremediation techniques. In this study, we sequenced, assembled, and analyzed the complete genome of Acinetobacter pittii CEP14, a strain isolated from chloroethene-contaminated groundwater, that has demonstrated the ability for aerobic cometabolic degradation of cDCE in the presence of n-hexane, phenol, and toluene. The A. pittii CEP14 genome consists of a 3.93 Mbp-long chromosome (GenBank accession no. CP084921) with a GC content of 38.9% and three plasmids (GenBank accession no. CP084922, CP084923, and CP084924). Gene function was assigned to 83.4% of the 3,930 coding DNA sequences. Functional annotation of the genome revealed that the CEP14 strain possessed all genetic elements to mediate the degradation of a range of aliphatic and aromatic compounds, including n-hexane and phenol. In addition, it harbors gene clusters involved in cytosol detoxification and oxidative stress resistance, which could play a role in the mitigation of toxic chemical intermediates that can arise during the degradation of cDCE. Gene clusters for heavy metal and antibiotic resistance were also identified in the genome of CEP14. These results suggest that CEP14 may be a versatile degrader of xenobiotic compounds and well-adapted to polluted environments, where a combination of heavy metal and organic compound pollution is often found.

Phenotypic and Genotypic Characteristics of a Tigecycline-Resistant Acinetobacter pittii Isolate Carrying bla NDM-1 and the Novel bla OXA Allelic Variant bla OXA-1045.

A tigecycline-resistant Acinetobacter pittii clinical strain from pleural fluid carrying a bla NDM-1 gene and a novel bla OXA gene, bla OXA-1045, was isolated and characterized. The AP2044 strain acquired two copies of the bla NDM-1 gene and six antibiotic resistance genes (ARGs) from other pathogens. According to the whole-genome investigation, the GC ratios of ARGs (50-60%) were greater than those of the chromosomal backbone (39.46%), indicating that ARGs were horizontally transferred. OXA-1045 belonged to the OXA-213 subfamily and the amino acid sequence of OXA-1045 showed 89% similarity to the amino acid sequences of OXA-213. Then, bla OXA-1045 and bla OXA-213 were cloned and the minimum inhibitory concentrations (MICs) of ß-lactams in the transformants were determined using the broth microdilution method. OXA-1045 was able to confer a reduced susceptibility to piperacillin and piperacillin-tazobactam compared to OXA-213. AP2044 strain exhibited low pathogenicity in Galleria mellonella infection models. The observation of condensed biofilm using the crystal violet staining method and scanning electron microscopy (SEM) suggested that the AP2044 strain was a weak biofilm producer. Quantitative reverse transcription-PCR (qRT-PCR) was used to detect the expression of resistance-nodulation-cell division (RND) efflux pump-related genes. The transcription level of adeB and adeJ genes increased significantly and was correlated with tigecycline resistance. Therefore, our genomic and phenotypic investigations revealed that the AP2044 strain had significant genome plasticity and natural transformation potential, and the emergence of antibiotic resistance in these unusual bacteria should be a concern for future investigations.

Genetic Resistance Determinants in Clinical Acinetobacter pittii Genomes.

Antimicrobial-resistant pathogenic bacteria are an increasing problem in public health, especially in the healthcare environment, where nosocomial infection microorganisms find their niche. Among these bacteria, the genus Acinetobacter which belongs to the ESKAPE pathogenic group harbors different multi-drug resistant (MDR) species that cause human nosocomial infections. Although A. baumannii has always attracted more interest, the close-related species A. pittii is the object of more study due to the increase in its isolation and MDR strains. In this work, we present the genomic analysis of five clinically isolated A. pittii strains from a Spanish hospital, with special attention to their genetic resistance determinants and plasmid structures. All the strains harbored different genes related to ß-lactam resistance, as well as different MDR efflux pumps. We also found and described, for the first time in this species, point mutations that seem linked with colistin resistance, which highlights the relevance of this comparative analysis among the pathogenic species isolates.

Emergence of ST63 Pandrug-Resistant Acinetobacter pittii Isolated From an AECOPD Patient in China.

Acinetobacter sp. is among the ESKAPE organisms which represent the major nosocomial pathogens that exhibited a high resistance rate. A. pittii, frequently associated with antimicrobial resistance particularly to carbapenems, is one of the most common Acinetobacter species causing invasive infection. Pandrug resistant A. pittii has rarely been reported. Here, we report the case of a patient with acute exacerbations of chronic obstructive pulmonary disease three years after double lung transplantation and developed severe pneumonia associated with pandrug resistant A. pittii infection. Phenotypic and genomic characteristics of this pandrug resistant isolate (17-84) was identified, and the mechanisms underlying its resistance phenotypes were analyzed. Isolate 17-84 belonged to ST63, carried a non-typable and non-transferable plasmid encoding multiple acquired resistance genes including carbapenemase gene blaOXA-58. Point mutations and acquired resistance genes were identified which were associated with different drug resistance phenotypes. To our knowledge, this is the first detailed phenotypic and genomic characterization of PDR A. pittii causing severe infections in clinical settings. Findings from us and others indicate that A. pittii could serve as a reservoir for carbapenem determinants. The emergence of such a superbug could pose a serious threat to public health. Further surveillance of PDR A. pittii strains and implementation of stricter control measures are needed to prevent this emerging pathogen from further disseminating in hospital settings and the community.

Simulated Microgravity Promotes Horizontal Gene Transfer of Antimicrobial Resistance Genes between Bacterial Genera in the Absence of Antibiotic Selective Pressure.

Bacteria are able to adapt and survive in harsh and changing environments through many mechanisms, with one of them being horizontal gene transfer (HGT). This process is one of the leading culprits in the spread of antimicrobial resistance (AMR) within bacterial communities and could pose a significant health threat to astronauts if they fell ill, especially on long-duration space missions. In order to better understand the degree of HGT activity that could occur in space, biosafety level-2, donor and recipient bacteria were co-cultured under simulated microgravity (SMG) on Earth with concomitant 1G controls. Two AMR genes, blaOXA-500 and ISAba1, from the donor Acinetobacter pittii, were tracked in four recipient strains of Staphylococcus aureus (which did not harbor those genes) using polymerase chain reaction. All four S. aureus strains that were co-cultured with A. pittii under SMG had a significantly higher number of isolates that were now blaOXA-500- and ISAba1-positive compared to growth at 1G. The acquisition of these genes by the recipient induced a phenotypic change, as these isolates were now resistant to oxacillin, which they were previously susceptible to. This is a novel study, presenting, for the first time, increased HGT activity under SMG and the potential impact of the space environment in promoting increased gene dissemination within bacterial communities.

Characterization and Transcriptome Analysis of a Long-Chain n-Alkane-Degrading Strain Acinetobacter pittii SW-1.

Strain sw-1, isolated from 7619-m seawater of the Mariana Trench, was identified as Acinetobacter pittii by 16S rRNA gene and whole-genome sequencing. A. pittii sw-1 was able to efficiently utilize long-chain n-alkanes (C18-C36), but not short- and medium-chain n-alkanes (C8-C16). The degradation rate of C20 was 91.25%, followed by C18, C22, C24, C32, and C36 with the degradation rates of 89.30%, 84.03%, 80.29%, 30.29%, and 13.37%, respectively. To investigate the degradation mechanisms of n-alkanes for this strain, the genome and the transcriptome analyses were performed. Four key alkane hydroxylase genes (alkB, almA, ladA1, and ladA2) were identified in the genome. Transcriptomes of strain sw-1 grown in C20 or CH3COONa (NaAc) as the sole carbon source were compared. The transcriptional levels of alkB and almA, respectively, increased 78.28- and 3.51-fold in C20 compared with NaAc, while ladA1 and ladA2 did not show obvious change. The expression levels of other genes involved in the synthesis of unsaturated fatty acids, permeases, membrane proteins, and sulfur metabolism were also upregulated, and they might be involved in n-alkane uptake. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) confirmed that alkB expression was significantly induced by C20, C24, and C32, and almA induction extent by C24 and C32 was higher than that with C20. Furthermore, ladA2 expression was only induced by C32, and ladA1 expression was not induced by any of n-alkanes. In addition, A. pittii sw-1 could grow with 0%-3% NaCl or 8 out of 10 kinds of the tested heavy metals and degrade n-alkanes at 15 °C. Taken together, these results provide comprehensive insights into the degradation of long-chain n-alkanes by Acinetobacter isolated from the deep ocean environment.

Diversity of Oxacillinases and Sequence Types in Carbapenem-Resistant Acinetobacter baumannii from Austria.

Carbapenem-resistant Acinetobacter baumannii is a significant health problem worldwide. A multicenter study on A. baumannii was performed to investigate the molecular epidemiology and genetic background of carbapenem resistance of A. baumannii isolates collected from 2014-2017 in Austria. In total, 117 non-repetitive Acinetobacter spp. assigned to A. baumannii (n = 114) and A. pittii (n = 3) were collected from four centers in Austria. The isolates were uniformly resistant to piperacillin/tazobactam, ceftazidime, and carbapenems, and resistance to imipenem and meropenem was 97.4% and 98.2%, respectively. The most prominent OXA-types were OXA-58-like (46.5%) and OXA-23-like (41.2%), followed by OXA-24-like (10.5%), with notable regional differences. Carbapenem-hydrolyzing class D carbapenemases (CHDLs) were the only carbapenemases found in A.baumannii isolates in Austria since no metallo-ß-lactamases (MBLs) nor KPC or GES carbapenemases were detected in any of the isolates. One-third of the isolates harbored multiple CHDLs. The population structure of A. baumannii isolates from Austria was found to be very diverse, while a total of twenty-three different sequence types (STs) were identified. The most frequent was ST195 found in 15.8%, followed by ST218 and ST231 equally found in 11.4% of isolates. Two new ST types, ST2025 and ST2026, were detected. In one A. pittii isolate, blaOXA-143-like was detected for the first time in Austria.

Genomic Characterization of Clinical Extensively Drug-Resistant Acinetobacter pittii Isolates.

Carbapenem-resistant Acinetobacter pittii (CRAP) is a causative agent of nosocomial infections. This study aimed to characterize clinical isolates of CRAP from a tertiary hospital in Northeast Thailand. Six isolates were confirmed as extensively drug-resistant Acinetobacter pittii (XDRAP). The blaNDM-1 gene was detected in three isolates, whereas blaIMP-14 and blaIMP-1 were detected in the others. Multilocus sequence typing with the Pasteur scheme revealed ST220 in two isolates, ST744 in two isolates, and ST63 and ST396 for the remaining two isolates, respectively. Genomic characterization revealed that six XDRAP genes contained antimicrobial resistance genes: ST63 (A436) and ST396 (A1) contained 10 antimicrobial resistance genes, ST220 (A984 and A864) and ST744 (A56 and A273) contained 9 and 8 antimicrobial resistance genes, respectively. The single nucleotide polymorphism (SNP) phylogenetic tree revealed that the isolates A984 and A864 were closely related to A. pittii YB-45 (ST220) from China, while A436 was related to A. pittii WCHAP100020, also from China. A273 and A56 isolates (ST744) were clustered together; these isolates were closely related to strains 2014S07-126, AP43, and WCHAP005069, which were isolated from Taiwan and China. Strict implementation of infection control based upon the framework of epidemiological analyses is essential to prevent outbreaks and contain the spread of the pathogen. Continued surveillance and close monitoring with molecular epidemiological tools are needed.

Phenotypic Variation and Carbapenem Resistance Potential in OXA-499-Producing Acinetobacter pittii.

Acinetobacter pittii is increasingly recognized as a clinically important species. Here, we identified a carbapenem-non-resistant A. pittii clinical isolate, A1254, harboring bla OXA- 499, bla OXA- 826, and bla ADC- 221. The bla OXA- 499 genetic environment in A1254 was identical to that of another OXA-499-producing, but carbapenem-resistant, A. pittii isolate, YMC2010/8/T346, indicating the existence of phenotypic variation among OXA-499-producing A. pittii strains. Under imipenem-selective pressure, the A1254 isolate developed resistance to carbapenems in 60 generations. Two carbapenem-resistant mutants (CAB009 and CAB010) with mutations in the bla OXA- 499 promoter region were isolated from two independently evolved populations (CAB001 and CAB004). The CAB009 mutant, with a mutation at position -14 (A to G), exhibited a four-fold higher carbapenem minimum inhibitory concentration (MIC) and a 4.53 ± 0.19 log2 fold change higher expression level of bla OXA- 499 than the ancestor strain, A1254. The other mutant, CAB010, with a mutation at position -42 (G to A), showed a two-fold higher carbapenem MIC and a 1.65 ± 0.25 log2 fold change higher bla OXA- 499 expression level than the ancestor strain. The bla OXA- 499 gene and its promoter region were amplified from the wild-type strain and two mutant isolates and then individually cloned into the pYMAb2-Hyg r vector and expressed in Acinetobacter baumannii ATCC 17978, A. pittii LMG 1035, and A. pittii A1254. All the transformed strains were resistant to carbapenem, irrespective of whether they harbored the initial or an evolved promoter sequence, and transformed strains expressing the promoter from the most resistant mutant, CAB009, showed the highest carbapenem MICs, with values of 32-64 µg/ml for imipenem and 128 µg/ml for meropenem. RNA sequencing was performed to confirm the contribution of bla OXA- 499 to the development of carbapenem resistance. Although the CAB009 and CAB010 transcriptional patterns were different, bla OXA- 499 was the only differentially expressed gene shared by the two mutants. Our results indicate that carbapenem-non-resistant Acinetobacter spp. strains carrying bla OXA genes have the potential to develop carbapenem resistance and need to be further investigated and monitored to prevent treatment failure due to the development of resistance.

Isolation and Characterization of Phosphorus Solubilizing Bacteria With Multiple Phosphorus Sources Utilizing Capability and Their Potential for Lead Immobilization in Soil.

Phosphorus solubilizing bacteria (PSB) can promote the level of plant-absorbable phosphorus (P) in agro-ecosystems. However, little attention has been paid to PSB harboring abilities in utilizing multiple phosphorus sources and their potentials for heavy metal immobilization. In this study, we applied the strategy of stepwise acclimation by using Ca3(PO4)2, phytate, FePO4, and AlPO4 as sole P source. We gained 18 PSB possessing abilities of multiple P sources utilization, and these bacteria belonged to eight genera (Acinetobacter, Pseudomonas, Massilia, Bacillus, Arthrobacter, Stenotrophomonas, Ochrobactrum, and Cupriavidus), and clustered to two apparent parts: Gram-positive bacteria and Gram-negative bacteria. The isolate of Acinetobacter pittii gp-1 presented good performance for utilizing Ca3(PO4)2, FePO4, AlPO4, and phytate, with corresponding P solubilizing levels were 250.77, 46.10, 81.99, and 7.91 mg/L PO4 3--P, respectively. The PSB A. pittii gp-1 exhibited good performance for solubilizing tricalcium phosphate in soil incubation experiments, with the highest values of water soluble P and available P were 0.80 and 1.64 mg/L, respectively. Additionally, the addition of A. pittii gp-1 could promote the immobilization of lead (Pb), and the highest Pb immobilization efficiency reached 23%. Simultaneously, we found the increases in abundances of both alkaline phosphatase gene (phoD) and ß-propeller phytase gene (bpp) in strain gp-1 added soils. Besides, we observed the expression up-regulation of both pyrroloquinoline quinone gene (pqq) and polyphosphate kinases gene (ppk), with the highest relative expression levels of 18.18 and 5.23, respectively. We also found the polyphosphate particles using granule staining. To our knowledge, our findings first suggest that the solubilizing of tricalcium phosphate by phosphorus solubilizing bacterium belonging to Acinetobacter is coupled with the synthesis of polyphosphate. Taken together, A. pittii gp-1 could be a good candidate in improving soil fertility and quality.

The characteristics and genome analysis of vB_ApiP_XC38, a novel phage infecting Acinetobacter pittii.

Acinetobacter pittii is an important pathogen causing nosocomial infection worldwide. In this study, a multidrug-resistant A. pittii ABC38 was used as host bacterium to isolate the lytic phage vB_ApiP_XC38. The biological characteristics of vB_ApiP_XC38 were studied and the genome was sequenced and analyzed. vB_ApiP_XC38 belonged to Podoviridae family. The phage had double-stranded genome, which comprised 79,328 bp with 39.58% G+C content displaying very low similarity (< 1% identity) with published genomes of other phages and bacteria. A total of 97 open reading frames (ORFs) were predicted and contained nucleotide metabolism and replication module, structural components module, and lysis module. The ANI, AAI, and phylogenetic analysis indicated that all phages were found distant from vB_ApiP_XC38. Altogether, morphological, genomics, and phylogenetic analysis suggest that vB_ApiP_XC38 is more likely a novel phage of A. pittii.