Heteroresistance to beta-lactam antibiotics may often be a stage in the progression to antibiotic resistance.

Antibiotic resistance is a growing crisis that threatens many aspects of modern healthcare. Dogma is that resistance often develops due to acquisition of a resistance gene or mutation and that when this occurs, all the cells in the bacterial population are phenotypically resistant. In contrast, heteroresistance (HR) is a form of antibiotic resistance where only a subset of cells within a bacterial population are resistant to a given drug. These resistant cells can rapidly replicate in the presence of the antibiotic and cause treatment failures. If and how HR and resistance are related is unclear. Using carbapenem-resistant Enterobacterales (CRE), we provide evidence that HR to beta-lactams develops over years of antibiotic usage and that it is gradually supplanted by resistance. This suggests the possibility that HR may often develop before resistance and frequently be a stage in its progression, potentially representing a major shift in our understanding of the evolution of antibiotic resistance.

Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States.

Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States.IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.

Absence of mgrB Alleviates Negative Growth Effects of Colistin Resistance in Enterobacter cloacae.

Colistin is an important last-line antibiotic to treat highly resistant Enterobacter infections. Resistance to colistin has emerged among clinical isolates but has been associated with a significant growth defect. Here, we describe a clinical Enterobacter isolate with a deletion of mgrB, a regulator of colistin resistance, leading to high-level resistance in the absence of a growth defect. The identification of a path to resistance unrestrained by growth defects suggests colistin resistance could become more common in Enterobacter.

Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control infection.

Antibiotic-resistant bacteria are a significant threat to human health, with one estimate suggesting they will cause 10 million worldwide deaths per year by 2050, surpassing deaths due to cancer1. Because new antibiotic development can take a decade or longer, it is imperative to effectively use currently available drugs. Antibiotic combination therapy offers promise for treating highly resistant bacterial infections, but the factors governing the sporadic efficacy of such regimens have remained unclear. Dogma suggests that antibiotics ineffective as monotherapy can be effective in combination2. Here, using carbapenem-resistant Enterobacteriaceae (CRE) clinical isolates, we reveal the underlying basis for the majority of effective combinations to be heteroresistance. Heteroresistance is a poorly understood mechanism of resistance reported for different classes of antibiotics3-6 in which only a subset of cells are phenotypically resistant7. Within an isolate, the subpopulations resistant to different antibiotics were distinct, and over 88% of CRE isolates exhibited heteroresistance to multiple antibiotics ('multiple heteroresistance'). Combinations targeting multiple heteroresistance were efficacious, whereas those targeting homogenous resistance were ineffective. Two pan-resistant Klebsiella isolates were eradicated by combinations targeting multiple heteroresistance, highlighting a rational strategy to identify effective combinations that employs existing antibiotics and could be clinically implemented immediately.

A Nationwide Screen of Carbapenem-Resistant Klebsiella pneumoniae Reveals an Isolate with Enhanced Virulence and Clinically Undetected Colistin Heteroresistance.

The convergence of hypervirulence and multidrug resistance in Klebsiella pneumoniae is a significant concern. Here, we report the first screen for hypermucoviscosity, a trait associated with increased virulence, using a U.S. surveillance collection of carbapenem-resistant (CR) K. pneumoniae isolates. We identified one hypermucoviscous isolate, which carried a gene encoding the KPC-3 carbapenemase, among numerous resistance genes. The strain further exhibited colistin heteroresistance undetected by diagnostics. This convergence of diverse resistance mechanisms and increased virulence underscores the need for enhanced K. pneumoniae surveillance.

Carbapenem-Resistant Klebsiella pneumoniae Exhibiting Clinically Undetected Colistin Heteroresistance Leads to Treatment Failure in a Murine Model of Infection.

Antibiotic resistance is a growing crisis and a grave threat to human health. It is projected that antibiotic-resistant infections will lead to 10 million annual deaths worldwide by the year 2050. Among the most significant threats are carbapenem-resistant Enterobacteriaceae (CRE), including carbapenem-resistant Klebsiella pneumoniae (CRKP), which lead to mortality rates as high as 40 to 50%. Few treatment options are available to treat CRKP, and the polymyxin antibiotic colistin is often the "last-line" therapy. However, resistance to colistin is increasing. Here, we identify multidrug-resistant, carbapenemase-positive CRKP isolates that were classified as susceptible to colistin by clinical diagnostics yet harbored a minor subpopulation of phenotypically resistant cells. Within these isolates, the resistant subpopulation became predominant after growth in the presence of colistin but returned to baseline levels after subsequent culture in antibiotic-free media. This indicates that the resistance was phenotypic, rather than due to a genetic mutation, consistent with heteroresistance. Importantly, colistin therapy was unable to rescue mice infected with the heteroresistant strains. These findings demonstrate that colistin heteroresistance may cause in vivo treatment failure during K. pneumoniae infection, threatening the use of colistin as a last-line treatment for CRKP. Furthermore, these data sound the alarm for use of caution in interpreting colistin susceptibility test results, as isolates identified as susceptible may in fact resist antibiotic therapy and lead to unexplained treatment failures.IMPORTANCE This is the first report of colistin-heteroresistant K. pneumoniae in the United States. Two distinct isolates each led to colistin treatment failure in an in vivo model of infection. The data are worrisome, especially since the colistin heteroresistance was not detected by current diagnostic tests. As these isolates were carbapenem resistant, clinicians might turn to colistin as a last-line therapy for infections caused by such strains, not knowing that they in fact harbor a resistant subpopulation of cells, potentially leading to treatment failure. Our findings warn that colistin susceptibility testing results may be unreliable due to undetected heteroresistance and highlight the need for more accurate and sensitive diagnostics.

Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae.

Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics.

Mechanisms of Antimicrobial Peptide Resistance in Gram-Negative Bacteria.

Cationic antimicrobial peptides (CAMPs) are important innate immune defenses that inhibit colonization by pathogens and contribute to clearance of infections. Gram-negative bacterial pathogens are a major target, yet many of them have evolved mechanisms to resist these antimicrobials. These resistance mechanisms can be critical contributors to bacterial virulence and are often crucial for survival within the host. Here, we summarize methods used by Gram-negative bacteria to resist CAMPs. Understanding these mechanisms may lead to new therapeutic strategies against pathogens with extensive CAMP resistance.

Induction of human plasmablasts during infection with antibiotic-resistant nosocomial bacteria.

OBJECTIVES: Nosocomial pathogens such as Acinetobacter baumannii are a growing public health threat, due in part to their increasing resistance to antibiotics. Since some strains are resistant to all available antibiotics, novel therapies are urgently needed. Plasmablasts are short-lived B cells found in the blood that can be collected and harnessed to produce therapeutic antibodies. We set out to determine whether plasmablasts are induced during infection with A. baumannii and other nosocomial pathogens. METHODS: We obtained blood samples from patients infected with antibiotic-resistant nosocomial pathogens, and analysed their plasmablast response by flow cytometry. RESULTS: We observed a strong induction of plasmablasts in patients with antibiotic-resistant A. baumannii infection. Furthermore, plasmablasts were also induced in response to other drug-resistant nosocomial pathogens. CONCLUSIONS: These data suggest that plasmablasts may be broadly harnessed to develop therapeutic antibodies to combat otherwise untreatable antibiotic-resistant infections.