Functional and Structural Characterization of OXA-935, a Novel OXA-10-Family ß-Lactamase from Pseudomonas aeruginosa.

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic ß-lactamase AmpC. However, OXA-10-family ß-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA ß-lactamases have a propensity for mutation that leads to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk P. aeruginosa clonal complex CC446 with a resistance to ceftazidime. A genomic analysis revealed that these isolates harbored a plasmid containing a novel allele of blaOXA-10, named blaOXA-935, which was predicted to produce an OXA-10 variant with two amino acid substitutions: an aspartic acid instead of a glycine at position 157 and a serine instead of a phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with the resistance of P. aeruginosa to ceftazidime. Compared to OXA-14, OXA-935 showed increased catalytic efficiency for ceftazidime. The deletion of blaOXA-935 restored the sensitivity to ceftazidime, and susceptibility profiling of P. aeruginosa laboratory strains expressing blaOXA-935 revealed that OXA-935 conferred ceftazidime resistance. To better understand the impacts of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. Compared to OXA-14, the F153S mutation in OXA-935 conferred increased flexibility in the omega (Ω) loop. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family ß-lactamases are concerning, given the rising reliance on novel ß-lactam/ß-lactamase inhibitor combinations, such as ceftolozane-tazobactam and ceftazidime-avibactam, to treat MDR P. aeruginosa infections.

Breakthrough daptomycin-, linezolid-, vancomycin-resistant Enterococcus faecium bacteremia during protracted daptomycin therapy: A case report.

Infections with multidrug resistant (MDR) Enterococcus faecium (Efm) are a growing problem. Vancomycin resistance in enterococci has long challenged treatment, necessitating the use of linezolid or daptomycin. Subsequently, daptomycin-, linezolid-, vancomycin-resistant Efm (DLVRE) infections have emerged. Case reports and guidelines for treating DLVRE infections are limited. Here, we describe the clinical and laboratory management of an MDR Efm protracted intraabdominal (IA) infection and breakthrough DLVRE bacteremia. Serial Efm resistance was evaluated using whole genome sequencing (WGS), susceptibility testing, and synergy analysis. Prior to in vitro synergy testing, combination antimicrobial therapy with daptomycin (DAP) and ceftaroline (CPT) was employed to treat the patient's central line-associated DLVRE bloodstream infection. In vitro antimicrobial testing revealed no synergy between daptomycin and ceftaroline; however, the patient's bacteremia cleared following initiation of both in conjunction with catheter removal. Sequencing of the DLVRE isolates revealed multiple genomic mutations which explained both linezolid and daptomycin resistance phenotypes and confirmed the presence of a plasmid containing the vanA operon. Sequential WGS of two additional bacterial isolates from the same patient revealed protracted colonization with a single DLVRE clone and suggested the development of bacterial subpopulations. Pairing clinical isolate susceptibilities with WGS and synergy testing should be encouraged in clinical practice to better inform antimicrobial management in cases of multidrug resistance.

Genomic surveillance for multidrug-resistant or hypervirulent Klebsiella pneumoniae among United States bloodstream isolates.

BACKGROUND: Klebsiella pneumoniae strains have been divided into two major categories: classical K. pneumoniae, which are frequently multidrug-resistant and cause hospital-acquired infections in patients with impaired defenses, and hypervirulent K. pneumoniae, which cause severe community-acquired and disseminated infections in normal hosts. Both types of infections may lead to bacteremia and are associated with significant morbidity and mortality. The relative burden of these two types of K. pneumoniae among bloodstream isolates within the United States is not well understood. METHODS: We evaluated consecutive K. pneumoniae isolates cultured from the blood of hospitalized patients at Northwestern Memorial Hospital (NMH) in Chicago, Illinois between April 2015 and April 2017. Bloodstream isolates underwent whole genome sequencing, and sequence types (STs), capsule loci (KLs), virulence genes, and antimicrobial resistance genes were identified in the genomes using the bioinformatic tools Kleborate and Kaptive. Patient demographic, comorbidity, and infection information, as well as the phenotypic antimicrobial resistance of the isolates were extracted from the electronic health record. Candidate hypervirulent isolates were tested in a murine model of pneumonia, and their plasmids were characterized using long-read sequencing. We also extracted STs, KLs, and virulence and antimicrobial resistance genes from the genomes of bloodstream isolates submitted from 33 United States institutions between 2007 and 2021 to the National Center for Biotechnology Information (NCBI) database. RESULTS: Consecutive K. pneumoniae bloodstream isolates (n = 104, one per patient) from NMH consisted of 75 distinct STs and 51 unique capsule loci. The majority of these isolates (n = 58, 55.8%) were susceptible to all tested antibiotics except ampicillin, but 17 (16.3%) were multidrug-resistant. A total of 32 (30.8%) of these isolates were STs of known high-risk clones, including ST258 and ST45. In particular, 18 (17.3%) were resistant to ceftriaxone (of which 17 harbored extended-spectrum beta-lactamase genes) and 9 (8.7%) were resistant to meropenem (all of which harbored a carbapenemase genes). Four (3.8%) of the 104 isolates were hypervirulent K. pneumoniae, as evidenced by hypermucoviscous phenotypes, high levels of virulence in a murine model of pneumonia, and the presence of large plasmids similar to characterized hypervirulence plasmids. These isolates were cultured from patients who had not recently traveled to Asia. Two of these hypervirulent isolates belonged to the well characterized ST23 lineage and one to the re-emerging ST66 lineage. Of particular concern, two of these isolates contained plasmids with tra conjugation loci suggesting the potential for transmission. We also analyzed 963 publicly available genomes of K. pneumoniae bloodstream isolates from locations within the United States. Of these, 465 (48.3%) and 760 (78.9%) contained extended-spectrum beta-lactamase genes or carbapenemase genes, respectively, suggesting a bias towards submission of antibiotic-resistant isolates. The known multidrug-resistant high-risk clones ST258 and ST307 were the predominant sequence types. A total of 32 (3.3%) of these isolates contained aerobactin biosynthesis genes and 26 (2.7%) contained at least two genetic features of hvKP strains, suggesting elevated levels of virulence. We identified 6 (0.6%) isolates that were STs associated with hvKP: ST23 (n = 4), ST380 (n = 1), and ST65 (n = 1). CONCLUSIONS: Examination of consecutive isolates from a single center demonstrated that multidrug-resistant high-risk clones are indeed common, but a small number of hypervirulent K. pneumoniae isolates were also observed in patients with no recent travel history to Asia, suggesting that these isolates are undergoing community spread in the United States. A larger collection of publicly available bloodstream isolate genomes also suggested that hypervirulent K. pneumoniae strains are present but rare in the USA; however, this collection appears to be heavily biased towards highly antibiotic-resistant isolates (and correspondingly away from hypervirulent isolates).

Family medicine in Iran: the birth of a new specialty.

Since the revolution of 1978-1979, the government of Iran has worked toward development of a primary health care system to improve basic health for its citizens. Although infant mortality and other parameters have improved, increasing urbanization and poor lifestyle choices continue to present major challenges to improving overall health statistics in the country. Generalist physicians, with no training beyond medical school graduation, have not inspired confidence from patients or specialist colleagues. Therefore, many patients prefer to receive care for common health complaints from specialist physicians. Health care for many individuals tends to be episodic, driven by patient concerns for acute illness rather than by patient-centered, longitudinal care. The government of Iran has decided to develop family medicine as a specialty within the country to help respond to these problems. Based on an initial consultation with some leaders in the Ministry of Health and Medical Education, as well as students, nursing staff, subspecialists, administrators, and medical educators, a number of suggested steps were recommended to support the development of family medicine in Iran. These involved, among others, further development of the specialty and parity with other specialties, development of faculty and curricula, and a plan for financing rural health care.

Distribution and ecology of meningeal worm, Parelaphostrongylus tenuis (Nematoda), in northcentral North America.

Meningeal worm (Parelaphostrongylus tenuis), a common nematode parasite in white-tailed deer (Odocoileus virginianus) and pathogenic for several species of ungulates in eastern North America, is not known to occur in the west. Heads of 1,902 white-tailed deer were examined for adult meningeal worm to determine geographic distribution of the parasite in Saskatchewan and Manitoba (Canada) and North Dakota (USA). Finding the parasite in a deer in eastern Saskatchewan near the Manitoba border established the current northern and western limits in Canada. Prevalence of infection was < 1, 18.6, and 8.2% in Saskatchewan, Manitoba, and North Dakota, respectively. Infected deer occurred throughout southern Manitoba and eastern North Dakota. Distribution appears to have changed little since the last published survey for P. tenuis in the region in 1972. We examined precipitation, temperature, deer density, and forest cover as likely correlates to prevalence and distribution of P. tenuis. Deer management units used for hunting purposes were the scale of analysis in the three jurisdictions. Presence of P. tenuis was positively correlated with precipitation during frost-free periods and deer density, and it was negatively correlated with winter and spring temperatures. Landscapes with > 25 and < 75% forest cover were most likely to have infected deer. Low rainfall and low density of white-tailed deer likely influence the westernmost limit of P. tenuis.

Climbing simulated vegetation to heights of ungulate hosts by larvae of Dermacentor albipictus (Acari: Ixodidae).

Larvae of winter ticks, Dermacentor albipictus (Packard), ascend vegetation in autumn and form clumps that attach to passing ungulate hosts. We tested the hypothesis that vegetation height determines the height of clumps. During the vegetation-to-ungulate transmission period (early September to mid-November), larvae were released at the base of simulated vegetation (nylon rods 245 cm tall) in outdoor and laboratory trials and in the absence of host cues. Rod height exceeded the height of the tallest ungulate host, which is the moose, Alces alces (L.). Most larvae stopped climbing and formed clumps 50-190 cm above ground, which coincided with torso heights of moose; elk, Cervus elaphus L.; and deer, Odocoileus spp. Rafinesque. More clumps formed in outdoor trials than in laboratory trials and clump heights tended to increase over the course of the experiment, but clump number, size, and height did not correlate with weather conditions. Winter tick larvae appear to determine their height above ground in the absence of external cues, but this mechanism may be modified by external conditions.

Meningeal worm evokes a heterogeneous immune response in elk.

Meningeal worm (Parelaphostrongylus tenuis) is a neurotropic nematode of ungulates in eastern North America. Lack of an effective diagnostic test increases the concern of translocating potentially infected ungulates into western North America, where P. tenuis does not occur naturally. In an attempt to identify serodiagnostic molecules, we determined (1) whether elk (Cervus elaphus) experimentally infected with P. tenuis produce antibodies against infective larvae or adult worms, and (2) if sera consistently recognize antigens that distinguish P. tenuis from a common nematode parasite of elk, the lungworm Dictyocaulus viviparus. Each of 10 elk were exposed to 15 or 300 infective P. tenuis larvae. Serum was collected (0, 41, and 83 days post-exposure and at necropsy) and monitored for antibodies using the enzyme-linked immunosorbent assay (ELISA) and immunoblot. When reactivity of sera with larval P. tenuis protein was compared (day 0 versus 83), ELISA values were significantly higher on day 83 for elk exposed to 15 or 300 parasites. Likewise, ELISA values using protein of adult P. tenuis were higher for elk exposed to 300 larvae. Immunoblots showed that sera from elk, with adult worms in the central nervous system, consistently recognized the 25-27, 28-30, and 34-36 kDa antigens of infective larvae after 83 days. However, many D. viviparus molecules were found to cross-react with antibodies formed against meningeal worm antigens. Use of adult worm proteins for serodiagnosis appears limited, because no protein was consistently recognized by sera collected from elk exposed to 15 larvae. We believe that development of a reliable diagnostic test for meningeal worm requires more research addressing cross-reactivity and detection of P. tenuis during the incubation stage.

Meningeal worm in experimentally-infected bighorn and domestic sheep.

In the first (July 1989) of two experiments, each of three bighorn sheep (Ovis canadensis) and three domestic sheep, respectively, was exposed to 25, 150, or 300 infective third-stage larvae (L3) of the meningeal worm, Parelaphostrongylus tenuis. Two bighorn sheep had temporary mild paresis and lumbar weakness; one developed paralysis and died suddenly 32 days after exposure. Adult P. tenuis were found deep within the brain and spinal cord of the one latter sheep. A generalized inflammatory response, characterized by subdural lymphoid aggregations adjacent to spinal nerve roots, was seen in the spinal cord of most domestic and bighorn sheep. In the second experiment (September 1990), each of six domestic sheep lambs and five white-tailed deer (Odocoileus virginianus) fawns was exposed to a single dose of 15 to 125 L3 of meningeal worm. Clinical signs were seen in only one sheep; it was dull and depressed. No worms were found in this sheep. One dead adult meningeal worm was found on the brain of another sheep. First-stage larvae and adult meningeal worms were found in all deer.

Antigens of adults and third-stage larvae of the meningeal worm, Parelaphostrongylus tenuis (Nematoda, Metastrongyloidea).

We studied the antigens of adults and third-stage larvae of the meningeal worm, Parelaphostrongylus tenuis, in an attempt to identify potential serodiagnostic molecules for this important infection of wild ungulates. Soluble extracts of P. tenuis adult worms and third-stage larvae were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional (2D) electrophoresis and analyzed by immunoblotting using purified rabbit anti-P. tenuis immunoglobulin G (IgG). The IgG antibodies were obtained from animals immunized with P. tenuis adult worm or third-stage larva soluble extract and serum from elk infected with P. tenuis. Out of more than 75 antigens (as shown by 2D electrophoresis and immunoblotting), 7 antigens from adults (four 170-120-kD molecules with isoelectric points between 6.0 and 6.6, two 55-kD molecules with isoelectric points of 5.6 and 5.8, and one 13-kD molecule) and 2 antigens from third-stage larvae (one 25-30-kD molecule with an isoelectric point of 6.3 and one 13-kD molecule) distinguished P. tenuis from two other nematodes, Dictyocaulus viviparus and Trichinella spiralis. Initial results using serum from experimentally infected elk indicate that this serum recognized a similar profile of P. tenuis antigens when compared with the serum from immunized rabbits. This research has set the foundation for the development of a test for P. tenuis infections in wild and recently domesticated elk and and other ungulates.

Mortality of fallow deer (Dama dama) experimentally-infected with meningeal worm, Parelaphostrongylus tenuis.

Six fallow deer (Dama dama) fawns died after receiving 25 to 150 infective larvae of Parelaphostrongylus tenuis. Fawns given higher doses usually died sooner (6 to 23 days) than those given lower doses (54 to 67 days). Early deaths were associated with severe acute peritonitis resulting from perforation of the intestinal wall; later deaths were associated with paralysis and inability to rise. Numerous adult P. tenuis were found within neural tissues of the brain and spinal cord in the three fawns with paralysis. One white-tailed deer (Odocoileus virginianus) exposed to infective larvae from the same source survived infection without exhibiting clinical signs and began passing larvae in feces 88 days post-exposure. At the doses used in this study, meningeal worm caused fatal infections in fallow deer. Results are compared to published observations of fallow deer naturally-infected with P. tenuis.

Ectoparasites from elk (Cervus elaphus nelsoni) from Wyoming.

Hides of nine elk, collected during the winter of 1986-1987 from the National Elk Refuge, Wyoming (USA) were examined for ectoparasites. Parasites recovered were mites, Psoroptes sp. (five elk); lice, Solenopotes ferrisi and Bovicola (Bovicola) longicornis (seven elk); and winter ticks, Dermacentor albipictus (nine elk). Three elk with severe scabies had an estimated 0.6 x 10(6), 3.8 x 10(6) and 6.5 x 10(6) mites, respectively. Densities of mites were much higher in skin regions with severe dermatitis. Skin lesions on elk with scabies consisted of dense, often moist, scabs extending along the dorsal and lateral thoracic regions of the body. Lesions attributed to winter ticks consisted of broken hair and alopecia on the dorsal portion of the lower neck, often extending in a "collar" around the neck.

Parelaphostrongylus andersoni (Nematoda: Protostrongylidae) in white-tailed deer from Michigan.

Dorsal-spined larvae in fecal samples from free-ranging white-tailed deer (Odocoileus virginianus) in Michigan and Pennsylvania were used as a source of larvae to infect a hand-raised white-tailed deer fawn. The fawn receive 200 third-stage larvae and passed dorsal-spined larvae in feces 66 days later. Muscleworm (Parelaphostrongylus andersoni), and meningeal worm (Parelaphostrongylus tenuis) were recovered at necropsy. Two white-tailed deer and seven wapiti (Cervus elaphus) exposed to larvae of the source from Pennsylvania harbored only P. tenuis. This is the first report of P. andersoni in the midwestern United States and extends the known range of this muscleworm in free-ranging white-tailed deer. Concurrent infections of P. andersoni and P. tenuis have not been established previously in experimentally infected fawns.

Bioenergetic consequences of alopecia induced by Dermacentor albipictus (Acari: Ixodidae) on moose.

Fasting metabolic rates, respiration rates, respiratory minute volumes, and fasted weights were measured on three yearling moose (Alces alces (L.)) (Artiodactyla: Cervidae) infested with 50,000 winter ticks. Dermacentor albipictus (Packard) (Acari: Ixodidae), and on two uninfested controls. Infestations produced no detectable effects on fasting metabolic rates or weight changes. The influence of tick-induced alopecia on lower critical temperatures could not be assessed because of warm temperatures during the winter and spring trials. Destruction of winter hair accompanied a reduction in respiratory minute volumes and respiration rates of heat-stressed moose.

Dermacentor albipictus (Acari, Ixodidae) on captive reindeer and free-ranging woodland caribou.

Infestations of winter ticks (Dermacentor albipictus) on two captive reindeer (Rangifer tarandus tarandus) are reported and may be associated with increased grooming and alopecia. Over 400,000 ticks were recovered from one reindeer. Few ticks (less than 25 ticks/animal) were found on three free-ranging woodland caribou (Rangifer tarandus caribou).

Factors influencing the potential establishment of the winter tick (Dermacentor albipictus) in Alaska.

The winter tick (Dermacentor albipictus) is not known to occur in Alaska. Survival and development of free-living (i.e., non-host-associated) stages of the tick were studied at three sites in central and southern Alaska. Female ticks survived, oviposited, and eggs hatched at all sites. Hatch success was low at one site where summer temperatures were low. Results suggest that establishment of winter ticks in Alaska following accidental translocation is possible, but several factors would affect such establishment.

Evaluation of random sampling for estimating density of winter ticks (Dermacentor albipictus) on moose (Alces alces) hides.

Densities of winter ticks (Dermacentor albipictus) were determined on each of 20 moose (Alces alces) half-hides by dissolving 100 cm2 quadrats in potassium hydroxide solution. Data were then used to determine the optimum sampling fraction for estimating tick densities. Random sampling was applied to 20 additional half-hides of known tick density to assess the accuracy of the estimates. We conclude that random sampling of 15% of the quadrats produces a good estimate of tick density. Total numbers of ticks were highly correlated with tick density.

Locations of moose in northwestern Canada with hair loss probably caused by the winter tick, Dermacentor albipictus (Acari: Ixodidae).

Five hundred two trappers representing 389 registered traplines in northern Alberta, northern British Columbia, Northwest Territories and Yukon Territory (Canada) responded to a questionnaire on the occurrence of hair loss and the winter tick (Dermacentor albipictus) on moose (Alces alces). Results suggested that winter ticks may occur as far as 62 degrees N. Several sightings of moose with presumed tick-induced hair loss near Kluane Lake, Yukon Territory, suggest the possibility of introduction of this serious pest into the moose population in Alaska.

Identification of dorsal-spined larvae from free-ranging wapiti (Cervus elaphus) in southwestern Manitoba, Canada.

Dorsal-spined first-stage larvae recovered from feces of free-ranging wapiti (Cervus elaphus) were passaged through snails (Triodopsis multilineata) and two hand-raised white-tailed deer fawns (Odocoileus virginianus). A total of 74 adult Parelaphostrongylus tenuis were recovered from the fawns; no other protostrongylid nematodes were recovered. The study indicates that wapiti may be infected with natural infections of meningeal worm and pass larvae suitable for transmission to gastropod intermediate hosts. Wapiti from areas endemic with P. tenuis should not be translocated to areas currently free of the parasite.

Instar development and disengagement rate of engorged female winter ticks, Dermacentor albipictus (Acari:Ixodidae), following single- and trickle-exposure of moose (Alces alces).

Seven hand-reared moose (Alces alces) calves and one yearling were infested with 30,000 larvae each of the winter tick. Dermacentor albipictus, either by single- or trickle-exposure (1000/day). They were examined weekly for instar changes from September/October until late May. By 2 and 3 weeks post-exposure, most larvae on single- and trickle-infested moose, respectively, had fed and molted to nymphs. Thereafter, tick development was similar between both infestation techniques. Nymphs dominated the tick population from October to mid-February, and adults from mid-February to May. The peak of host disengagement by engorged females was late March in both years. Weights of engorged females from calf moose declined over time during the disengagement period.

Effect of Dermacentor albipictus (Acari:Ixodidae) on blood composition, weight gain and hair coat of moose, Alces alces.

The physiological effects of the winter tick, Dermacentor albipictus, on moose, Alces alces, were investigated. Blood composition, weight gain, food intake and change in the hair coat of moose calves, four infested with D. albipictus larvae, and eight uninfested, were monitored. Infested moose groomed extensively, apparently in response to feeding nymphal and adult ticks, and developed alopecia. Other clinical signs included: chronic weight loss, anemia, hypoalbuminemia, hypophosphatemia, and transient decreases in serum aspartate transaminase and calcium during the period of nymphal and adult female tick engorgement. Infested animals did not become anorexic. Two moose with severe hair loss had increases in gamma globulin shortly after the onset of female tick engorgement. Results suggest that alopecia is associated with tick resistance. Animals that groom and develop hair loss likely carry fewer ticks and therefore suffer less severely from blood loss.