R93P Substitution in the PmrB HAMP Domain Contributes to Colistin Heteroresistance in Escherichia coli Isolates from Swine.

Here, the mechanisms of colistin heteroresistance (CHR) were assessed in 12 Escherichia coli isolates from swine in China. CHR was investigated by population analysis profile tests. CHR stability was studied by culturing isolates for five overnight incubation periods in colistin-free medium. Subsequently, the mcr-1 gene and mutations in PmrAB, PhoPQ, and MgrB were screened in parental isolates and resistant subpopulations. Additionally, the expression levels of phoPQ, its target gene pagP, and its negative regulator gene mgrB, as well as pmrAB and its target genes pmrHFIJKLM and pmrC, were determined by real-time relative quantitative PCR. Eleven of the 12 isolates were confirmed to show CHR, with 17 resistant subpopulations. Also, 11 of the 17 subpopulations (64.71%) harbored point mutations in PmrB and/or PhoQ, differing from their parental isolates. However, only one stable resistant subpopulation (EPF42-4) was identified; it harbored an arginine-to-proline substitution at position 93 (R93P) within the PmrB HAMP (histidine kinase, adenylyl cyclase, methyl-binding protein, and phosphatase) domain. Compared to the pmrB expression levels in the parental isolate EPF42 and E. coli K-12 MG1655, remarkable pmrB overexpression was observed in EPF42-4, which showed upregulated pmrA, pmrK, and pmrC expression. Structural analysis demonstrated that the R93P substitution promotes conformational changes in the HAMP domain, leading to an acceleration in its signal transduction ability and the activation of PmrB expression. In conclusion, point mutations in PmrB and/or PhoQ were primarily associated with CHR. The R93P substitution resulted in the establishment of stable resistant subpopulations in E. coli showing CHR.

Colistin heteroresistance in carbapenem-resistant Acinetobacter baumannii clinical isolates from a Thai university hospital.

Colistin is the last resort for the treatment of infections with carbapenem-resistant (CR) Gram-negative bacteria particularly Acinetobacter baumannii (CRAB). Currently, both colistin-resistant and -heteroresistant A. baumannii isolates have been reported globally. We therefore investigated the colistin heteroresistance rate in 75 non-duplicate colistin-susceptible CRAB clinical isolates from a Thai university collected in 2016. Minimum inhibitory concentrations (MICs) of colistin for all isolates were determined by broth microdilution method and carbapenemase genes were detected by PCR methods. All isolates were genotyped by ERIC-PCR method and screened for colistin heteroresistance by modified population analysis profile (PAP) method. The colistin MIC range for the 75 isolates was 0.5-2 µg/mL, with MIC50 and MIC90 of 1 and 2 µg/mL, respectively. Thirty-three isolates (44%) were considered colistin-heteroresistant with subpopulations growing at 3-8 µg/mL of colistin. After three daily passages of the subpopulations on antibiotic-free medium, their colistin MICs ranged from 4 to > 32 µg/mL, with MIC50 and MIC90 of 32 and > 32 µg/mL, respectively. Eight different ERIC-PCR profiles were obtained among the 33 isolates and all carried blaOXA-23-like. The high rate of colistin heteroresistance in the CRAB isolates highlights the possibility of treatment failure of CRAB infections by colistin due to the selection of colistin-resistant subpopulations.

Emergence of a carbapenem-resistant and colistin-heteroresistant Enterobacter cloacae clinical isolate in Japan.

A carbapenem-resistant and colistin-heteroresistant clinical isolate of Enterobacter cloacae was obtained from an inpatient in Okinawa, Japan. The minimum inhibitory concentrations of both imipenem and meropenem were 32 µg/mL. The isolate showed heteroresistance to colistin using the Etest method and resistance to colistin using the broth microdilution method. It had a disrupted ompC and a mutation in the promoter region of blaACT-2, but did not harbor any genes encoding carbapenemase. The disruption of ompC and the mutation in blaACT-2 was associated with the carbapenem resistance of this isolate. This isolate also had mutations in pmrAB and phoPQ encoding two-component regulatory systems, which may be associated with colistin heteroresistance.

Characterization of Carbapenemase-Producing Klebsiella pneumoniae Isolates from Two Romanian Hospitals Co-Presenting Resistance and Heteroresistance to Colistin.

Klebsiella pneumoniae is a notorious human pathogen involved in healthcare-associated infections. The worldwide expansion of infections induced by colistin-resistant and carbapenemase-producing Enterobacterales (CPE) isolates has been increasingly reported. This study aims to analyze the phenotypic and molecular profiles of 10 colistin-resistant (CR) isolates and 2 pairs of colistin-heteroresistant (ChR) (parental and the corresponding resistant mutants) isolates of K. pneumoniae CPE sourced from two hospitals. The phenotypes of strains in the selected collection had been previously characterized. Antimicrobial susceptibility testing was performed using a Vitek 2 Compact system (BioMérieux SA, Marcy l'Etoile, France), the disc diffusion method, and broth microdilution (BMD) for colistin. Whole-genome sequencing (WGS) did not uncover evidence of any mobile colistin resistance (mcr) genes, although the mgrB gene of seven isolates appeared to be disrupted by insertion sequences (ISKpn25 or ISKpn26). Possible deleterious missense mutations were found in phoP (L4F), phoQ (Q426L, L26Q, L224Q, Q317K), pmrB (R256G, P95L, T157P, V352E), and crrB (P151S) genes. The identified isolates belonged to the following clonal lineages: ST101 (n = 6), ST147 (n = 5), ST258 (n = 2), and ST307 (n = 1). All strains harbored IncF plasmids. OXA-48 producers carried IncL and IncR plasmids, while one blaNDM-1 genome was found to harbor IncC plasmids. Ceftazidime-avibactam remains a therapeutic option for KPC-2 and OXA-48 producers. Resistance to meropenem-vaborbactam has emerged in some blakPC-2-carrying isolates. Our study demonstrates that the results of WGS can provide essential evidence for the surveillance of antimicrobial resistance.

Colistin Dependency among Colistin-Heteroresistant Acinetobacter baumannii Isolates.

Colistin dependent (CD) isolates are dependent on colistin for optimal growth. Here we aimed to systematically determine the emergence of CD among colistin-heteroresistant carbapenem-resistant Acinetobacter baumannii (CRAB) isolates. We also examined the phenotypic characteristics of CD and the evolution of CD strains to overt resistance. Additionally, we examined whether detection of growth in blood cultures was impaired by CD. Heteroresistant isolates, as determined by population analysis profiling, were exposed to colistin; when the colony count with colistin was significantly higher than without, isolates were suspected to be CD. CD was confirmed by Etest and growth curves. CD strains with colistin minimum inhibitory concentrations > 2 mg/L after growth in colistin-free media were considered colistin-resistant. Of the 65 heteroresistant strains tested, eight became CD after colistin exposure. These strains attained higher colony counts and growth rates with colistin vs. without, and grew adjacent to the colistin Etest strip. CD strains exhibited increased susceptibilities to multiple antibiotics compared to their parent heteroresistant strains. All CD strains tested became colistin-resistant following growth without colistin. CD strains were detected in blood culture bottles, but time to detection was significantly prolonged compared with parent strains, suggesting that CD may lead to delay in detection of CRAB bacteremia.

Colistin Heteroresistance among Extended Spectrum ß-lactamases-Producing Klebsiella pneumoniae.

Colistin-heteroresistant (CST-HR) Enterobacterales isolates have been identified recently, challenging the clinical laboratories since routine susceptibility tests fail to detect this phenotype. In this work we describe the first CST-HR phenotype in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae isolates in South America. Additionally, we determine the genomic mechanisms of colistin heteroresistance in these strains. The CST-HR phenotype was analyzed by the population analysis profile (PAP) method, and mutations associated with this phenotype were determined by whole-genome sequencing (WGS) and the local BLAST+ DB tool. As a result, 8/60 isolates were classified as CST-HR according to the PAP method. From WGS, we determined that the CST-HR isolates belong to three different Sequence Types (STs) and four K-loci: ST11 (KL15 and KL81), ST25 (KL2), and ST1161 (KL19). We identified diverse mutations in the two-component regulatory systems PmrAB and PhoPQ, as well as a disruption of the mgrB global regulator mediated by IS1-like and IS-5-like elements, which could confer resistance to CST in CST-HR and ESBL-producing isolates. These are the first descriptions in Chile of CST-HR in ESBL-producing K. pneumoniae isolates. The emergence of these isolates could have a major impact on the effectiveness of colistin as a "last resort" against these isolates, thus jeopardizing current antibiotic alternatives; therefore, it is important to consider the epidemiology of the CST-HR phenotype.

Detection of colistin heteroresistance in Acinetobacter baumannii from blood and respiratory isolates.

Polymyxins are one of the last-line antibiotics for multidrug-resistant Acinetobacter baumannii. Reports have demonstrated the emergence of colistin heteroresistance in A. baumannii, which can complicate assessment of minimum inhibitory concentrations and promote resistance to colistin. We aimed to determine the presence of colistin heteroresistance in A. baumannii isolates and correlate the results with clinical and microbiological outcomes via a retrospective study of 24 adult patients: 12 blood and 12 invasive respiratory cultures positive for colistin-susceptible A. baumannii between 1 January 2013 and 31 July 2015. Heteroresistance testing was performed by plating a 100-µL bacterial cell suspension on Mueller-Hinton agar plates containing 0, 1, 2, and 4 µg/mL colistin, and assessing for growth at 24 and 48 h. Colistin heteroresistance was exhibited in 83% of isolates. Median age was 56 [43-65] years, 10 (42%) patients resided at a facility prior to admission, 5 (21%) had a chronic tracheostomy, 18 (75%) were in the intensive care unit at the time of culture collection, and median infection-related length of stay was 12 [7-15] days. Clinical and microbiological cures were achieved in 75% of patients. Overall infection-related mortality was 21%. Our study demonstrated a high rate of colistin heteroresistance in clinical isolates of colistin-susceptible A. baumannii, although this was not associated with suboptimal clinical outcomes due to the use of aggressive colistin dosing and combination therapy. Further studies are needed to establish the association between in vitro colistin heteroresistance and clinical and microbiological outcomes.

Trends in the resistance profiles of Acinetobacter baumannii endemic clones in a university hospital of Argentina.

A total of 925 Acinetobacter spp. isolates were collected from routine clinical samples of patients admitted to the university hospital of Buenos Aires city during the period 2004-2012. From this collection, 129 isolates identified as Acinetobacter baumannii were selected for molecular studies. Minimal inhibitory concentrations (MICs) of antimicrobials were determined by agar dilution method. Colistin (COL) heteroresistance was investigated by means of population analysis studies. PCR-based methods were used for epidemiological analysis and for the screening of carbapenemases and the bla(tetB) gene. We have observed a steady rise in the MIC50 of imipenem (IMI)-resistant isolates and an increment in the presence of bla(OXA-23)-like gene (74-100%) as well. A rapid increasing rate of minocycline (MIN) resistance and a rise of the MIC50 of the resistant isolates have been detected since the year 2008. All isolates harboured the tet (B) gene. An increase in the value of the tigecycline (TIG) MIC was seen from the year 2007 onwards. This loss of activity was observed among different clones. A rise of COL heteroresistance from 46.4% in 2004 to 95% in 2012 was detected. During this period, COL consumption also increased (11.1-fold). However, COL resistance remained sporadic.