Phage resistance in Klebsiella pneumoniae and bidirectional effects impacting antibiotic susceptibility.

OBJECTIVES: Bacteriophage (phage) therapy is a promising anti-infective option to combat antimicrobial resistance. However, the clinical utilization of phage therapy has been severely compromised by the potential emergence of phage resistance. Although certain phage resistance mechanisms can restore bacterial susceptibility to certain antibiotics, a lack of knowledge of phage resistance mechanisms hinders optimal use of phages and their combination with antibiotics. METHODS: Genome-wide transposon screening was performed with a mutant library of Klebsiella pneumoniae MKP103 to identify phage pKMKP103_1-resistant mutants. Phage-resistant phenotypes were evaluated by time-kill kinetics and efficiency of plating assays. Phage resistance mechanisms were investigated with adsorption, one-step growth, and mutation frequency assays. Antibiotic susceptibility was determined with broth microdilution and population analysis profiles. RESULTS: We observed a repertoire of phage resistance mechanisms in K pneumoniae, such as disruption of phage binding (fhuA::Tn and tonB::Tn), extension of the phage latent period (mnmE::Tn and rpoN::Tn), and increased mutation frequency (mutS::Tn and mutL::Tn). Notably, in contrast to the prevailing view that phage resistance re-sensitizes antibiotic-resistant bacteria, we observed a bidirectional steering effect on bacterial antibiotic susceptibility. Specifically, rpoN::Tn increased susceptibility to colistin while mutS::Tn and mutL::Tn increased resistance to rifampicin and colistin. DISCUSSION: Our findings demonstrate that K pneumoniae employs multiple strategies to overcome phage infection, which may result in enhanced or reduced antibiotic susceptibility. Mechanism-guided phage steering should be incorporated into phage therapy to better inform clinical decisions on phage-antibiotic combinations.

A simple solid media assay for detection of synergy between bacteriophages and antibiotics.

The emergence of antibiotic-resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. Although phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation, and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat vancomycin-resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.IMPORTANCEBacteriophages have become an important alternative treatment for individuals with life-threatening antibiotic-resistant bacteria (ARB) infections. Because antibiotics represent the standard-of-care for treatment of ARB, antibiotics and phages often are delivered together without evidence that they work cooperatively. Testing for cooperativity can be difficult due to the equipment necessary and a lack of standardized means for performing the testing in liquid medium. We developed an assay using solid medium to identify interactions between antibiotics and phages for gram-positive and gram-negative bacteria. We modeled the interactions between antibiotics and phages on solid medium, and then tested multiple replicates of vancomycin-resistant Enterococcus (VRE) and Stenotrophomonas in the assay. For each organism, we identified synergy between different phage and antibiotic combinations. The development of this solid media assay for assessing synergy between phages and antibiotics will better inform the use of these combinations in the treatment of ARB infections.

Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

A Cross Sectional Study on the Bidirectional Interactions Between Leptospirosis and HIV Infection Among Patients from Maputo Central Hospital, Mozambique.

Introduction: This study aims to determine the baseline seroprevalence of leptospirosis, a zoonotic and neglected disease, in people living with HIV (PWH) in Maputo, Mozambique, and to evaluate the relationship between selected HIV-related factors that might influence risk of coinfection with leptospirosis, such as degree of immunosuppression, as assessed by CD4 cell count, World Health Organization (WHO) HIV/AIDS clinical stage and antiretroviral therapy (ART) intake. Methods: This was a descriptive cross-sectional analysis of 157 PWH, aged over 18 years old, admitted to the Maputo Central Hospital, in Maputo, Mozambique, between March 2020 and October 2021. The study participants were recruited as a convenience sample regardless of the reasons for their admission. We collected sociodemographic and clinical data, including ART and WHO HIV/AIDS clinical stage, and blood for CD4 cell count and detection of Leptospira IgG antibodies using a commercial Kit ab247199 Leptospira IgG ELISA (www.abcam.com/ab247199) with sensitivity and specificity of 100% and 97.3%, respectively. Laboratory testing was performed at the Faculty of Medicine, Eduardo Mondlane University and Laboratory of Clinical Analysis, in Maputo. Results: Participants were aged 18 to 72 years (median age 39 years; SD ± 10.5), the majority were female 100 (63.7%), from urban areas 138 (87.9%), with secondary-level education 80 (51%). The overall seroprevalence of Leptospira IgG antibodies was 40.1%. The median CD4 cell count was 385 cells/µl (02 to 2297; SD ± 378.47). Higher seroprevalence of Leptospira antibodies was found among participants with CD4 cell counts <250 cells/µl (54.8%), WHO HIV/AIDS stage IV (70.2%) and those on ART (92%), though there were no statistically significant differences between groups with and without Leptospira antibodies. Conclusion: Our study confirmed that Leptospira antibodies are highly prevalent in PWH in Maputo; however, Leptospira infection was not associated with the degree of immunosuppression, WHO HIV/AIDS clinical stage, or the use of ART. Our data support the need for routine screening for leptospirosis in PWH in Mozambique. Future studies are warranted to characterize the incidence and outcomes of symptomatic leptospirosis in this patient population and to identify circulating serovars and species in the country and region, as well as the implicated reservoirs.

Neurocysticercosis in Epileptic Children: An Overlooked Condition in Mozambique, Challenges in Diagnosis, Management and Research Priorities

Previous studies suggest that neurocysticercosis (NCC), the most common cause of acute symptomatic seizures (ASS), epilepsy and other neuropsychiatric disorders, typically presents with a solitary lesion and focal seizures in children from places where cysticercosis is endemic. We report a series of 3 patients, aged 7 to 11 years, with a history of epilepsy and or recurrent headache referred from Mocuba to the Quaternary Central Hospital in Quelimane, Zambeze Province, Mozambique, an area endemic for cysticercosis. Clinical history and examination, blood chemistry and hemogram screening, serological testing for Cysticercus antigens and antibodies detection, and a computerized tomography (CT) scan, were performed. NCC was confirmed in all 3 patients, based on criteria defined by Del Bruto. Two confirmed cases tested positive for antigen (Ag) by enzyme-linked immunosorbent assay (ELISA) with CT lesions in different stages of parasite evolution. Headache/encephalopathy was present in all patients. This case series of children with epilepsy confirms for the first time the presence of NCC in children from Zambezia province, an east-central region of Mozambique. Further, NCC should be included in the differential diagnosis of children with ASS, epilepsy and other neuropsychiatric disorders. Future studies should be targeted to the identification of biomarkers to support the diagnosis of NCC, given the limited availability of imaging tools and limited value of serological assays for the diagnosis and management of NCC.

Decreased memory T-cell response and function in human immunodeficiency virus-infected patients with tegumentary leishmaniasis

The effects of human immunodeficiency virus (HIV) on the immune response in patients with cutaneous leishmaniasis have not yet been fully delineated. This study quantified and evaluated the function of memory T-cell subsets in response to soluble Leishmania antigens (SLA) from patients coinfected with HIV and Leishmania with tegumentary leishmaniasis (TL). Eight TL/HIV coinfected subjects and 10 HIV seronegative subjects with TL were evaluated. The proliferative response of CD4+and CD8+T-cells and naïve, central memory (CM) and effector memory (EM) CD4+T-cells in response to SLA were quantified using flow cytometry. The median cell division indices for CD4+and CD8+T-cells of coinfected patients in response to SLA were significantly lower than those in patients with Leishmania monoinfection (p < 0.05). The proportions of CM and EM CD4+T-cells in response to SLA were similar between the coinfected patients and patients with Leishmania monoinfection. However, the median CM and EM CD4+T-cell counts from coinfected patients were significantly lower (p < 0.05). The reduction in the lymphoproliferative response to Leishmania antigens coincides with the decrease in the absolute numbers of both EM and CM CD4+T-cells in response to Leishmania antigens in patients coinfected with HIV/Leishmania.

A cross-sectional study of sub-clinical Plasmodium falciparum infection in HIV-1 infected and uninfected populations in Mozambique, South-Eastern Africa

Background: Plasmodium falciparum and HIV-1 infection cause substantial morbidity and mortality in sub-Saharan Africa. Increasing evidence suggests these two pathogens interact negatively when infecting the same individual. Methods: A cross-sectional study among HIV-1 infected and uninfected populations was recruited in Mocuba and Maputo, Mozambique to determine the prevalence of sub-clinical malarial parasitaemia using light microscopy and a nested PCR assay. Results: The prevalence of sub-clinical P. falciparum parasitaemia was low in Maputo, whether determined by microscopy (0.4%) or PCR (1.9%), but substantially higher in Mocuba (7.6 and 14.7%, respectively). Nested PCR detected nearly 70% more cases of sub-clinical parasitaemia than microscopy, but differences occur by locality. HIV-1 infected persons were more likely to be sub-clinically parasitaemic than HIV-1 uninfected individuals recruited from the same geographic areas. Trimethoprim-sulphamethoxazole use did not substantially reduce sub-clinical parasitaemia. Conclusions: Dried blood spots are a convenient and sensitive technique for detecting sub-clinical infection with P. falciparum by nested PCR. Prevalence of P. falciparum is substantially lower in Maputo where malaria control programmes have been more active than in the rural town of Mocuba. In Mocuba, among those presenting for HIV-1 counseling and testing, the prevalence of P. falciparum is substantially higher in those who test positive for HIV-1 than those without HIV-1 infection. The clinical implications of sub-clinical P. falciparum infection among HIV-1 infected persons warrant additional study.

An adherence trilogy is essential for long-term HAART success

Adherence is the milestone of a successful therapy. Over the last decade several authors have addressed the importance of adherence for optimal results of antiretroviral (ARV) therapy. Many health care systems are investing substantial resources to make available contemporary antiretroviral therapy. Despite the large investment in medications, insufficient investments have been made into an integrated adherence component to maximize the impact of these medications. Adherence, unlike drug therapy, cannot be defined as a single method with a defined prescription or formula. Instead, it is the result of a complex interaction between the patient, a prescribed medication and the health system. Many reports are available analyzing each of these components. We have found that critical elements of adherence include the patient's knowledge about the disease and how medications will help achieve a longer and healthier life, together with the motivation to adapt to a new style of life. A trilogy composed of information, motivation and behavioral skills is essential to achieve the maximum desired level of adherence. We have computerized this trilogy in a software program for self-administration in which each of the three components is provided to the patient as many times as necessary to transmit an understanding of the problem and to help make a rational decision to adhere to the ARV treatment program. In this review we analyze several efforts and techniques to improve adherence to any recommended medication that may interfere with the patient's lifestyle and outline how the adherence trilogy can be best used to optimize the ability of ARV therapy to durably suppress plasma HIV RNA to undetectable levels.