SARS-CoV-2 mutations on diagnostic gene targets in the second wave in Zimbabwe: A retrospective genomic analysis.

BACKGROUND: SARS-CoV-2 continues to be a major issue in resource-limited settings, particularly owing to the limited supply of vaccinescaused by inequitable distribution. OBJECTIVE: To monitor diagnostic gene targets to identify potential test failures caused by mutations, which is important for public health. METHODS: Here we analysed the genome sequence of SARS-CoV-2 from the second wave in Zimbabwe. A total of 377 samples weresequenced at Quadram Institute Bioscience. After quality control, 192 sequences passed and were analysed. RESULTS: The Beta variant was dominant during this period, contributing 77.6% (149) of the genomes sequenced and having a total of 2994mutations in diagnostic polymerase chain reaction target genes. Many single nucleotide polymorphism mutations resulted in amino acidsubstitution that had the potential to impact viral fitness by increasing the rate of transmission or evading the immune response to previousinfection or vaccination. CONCLUSION: There were nine lineages circulating in Zimbabwe during the second wave. The B.1.351 was dominant, accounting for >75%.There were over 3 000 mutations on the diagnostic genes and lineage B.1.351, contributing almost two-thirds of the mutations. The S-genehad the most mutations and the E-gene was the least mutated.

Design of clone-specific probes from genome sequences for rapid PCR-typing of outbreak pathogens.

The genome sequence of one OXA-48-producing Klebsiella pneumoniae belonging to sequence type (ST) 405, and three belonging to ST11, were used to design and test ST-specific PCR assays for typing OXA-48-producing K. pneumoniae. The approach proved to be useful for in-house development of rapid PCR typing assays for local outbreak surveillance.

Verbal numerical scales are as reliable and sensitive as visual analog scales for rating dyspnea in young and older subjects.

This study compared the use of a simple verbal 0-10 numerical rating scale (verbal NRS) and a visual analog scale (VAS) for the rating of dyspnea during exercise in a group of young and older subjects. Twelve younger (32+/-9 yr) and 12 older (71+/-7 yr) subjects used either the verbal NRS or the VAS in a randomised fashion to rate dyspnea during 60 s of uphill treadmill walking (range 5.6-8.8 km h(-1)) performed at either a low (17% grade) or high workload (26% grade) and then during recovery. Rating scales were evaluated twice on separate days (day 1 and day 2) at each workload. While the verbal NRS scores proved to be reliable throughout exercise and recovery, VAS scores were significantly (p<0.05) lower on day 2 during the low workload test (younger group) and the high workload test (older group). Verbal NRS ratings were consistently greater than VAS ratings at both workloads (p<0.001) for both young and older groups. The intra-class correlation coefficients for rating peak dyspnea using either the VAS or verbal NRS were consistently lower for the older subjects (range: r=0.54-0.67) than the younger subjects (range: r=0.70-0.86). Overall, subjects preferred the verbal NRS to the VAS. These results suggest that the verbal NRS compares favourably with the VAS for rating dyspnea during exercise without mask or mouthpiece. However, when rating peak dyspnea both scales appear less reliable when used by the older compared to young subjects.

Continuous and intermittent exercise responses in individuals with chronic obstructive pulmonary disease.

BACKGROUND: While the acute physiological responses to continuous exercise have been well documented in individuals with chronic obstructive pulmonary disease (COPD), no previous study has examined the response to intermittent exercise in these patients. METHODS: We examined the physiological responses of 10 individuals with moderate COPD (forced expiratory volume in 1 second 52 (15)% predicted) who performed both an intermittent (1 min exercise and rest intervals) and a continuous cycle ergometer test on separate days. Both intermittent and continuous exercise tests were performed at the same power output, calculated as 70% of the peak power attained during an incremental exercise test. RESULTS: Intermittent exercise was associated with significantly lower values for oxygen uptake, carbon dioxide output, expired ventilation, heart rate, plasma lactate concentration, and ratings of breathlessness than continuous exercise. Subjects were able to complete a significantly greater total amount of work during intermittent exercise (71 (32) kJ) than during continuous exercise (31 (24) kJ). The degree of dynamic lung hyperinflation (change in end expiratory lung volume) was significantly lower during intermittent exercise (0.23 (0.07) l) than in continuous exercise (0.52 (0.13) l). CONCLUSIONS: The greater amount of work performed and lower measured physiological responses achieved with intermittent exercise may allow for greater peripheral training adaptations in individuals with more limited lung function. The results suggest that intermittent exercise may be superior to continuous exercise as a mode of training for patients with COPD.

Animal models of Salmonella infections: enteritis versus typhoid fever.

The most common disease syndromes caused by Salmonella serotypes in humans, typhoid fever and enteritis, can be modeled using Salmonella enterica serotype Typhimurium infections in mice and calves, respectively. This article reviews murine typhoid and bovine enteritis and discusses strengths, limitations and distinctive features of these animal models.

Competitive exclusion of Salmonella enteritidis by Salmonella gallinarum in poultry.

Salmonella Enteritidis emerged as a major egg-associated pathogen in the late 20th century. Epidemiologic data from England, Wales, and the United States indicate that S. Enteritidis filled the ecologic niche vacated by eradication of S. Gallinarum from poultry, leading to an epidemic increase in human infections. We tested this hypothesis by retrospective analysis of epidemiologic surveys in Germany and demonstrated that the number of human S. Enteritidis cases is inversely related to the prevalence of S. Gallinarum in poultry. Mathematical models combining epidemiology with population biology suggest that S. Gallinarum competitively excluded S. Enteritidis from poultry flocks early in the 20th century.

Resuscitation by ferrioxamine E of stressed Salmonella enterica serovar typhimurium from soil and water microcosms.

Storage of Salmonella enterica serovar Typhimurium strains in soil and water microcosms resulted in loss of culturability on standard plating media. Prior incubation in buffered peptone water supplemented with ferrioxamine E markedly extended the time that bacteria were recoverable by plating, except in the case of mutants deficient in ferrioxamine E uptake.

Host adaptation and the emergence of infectious disease: the Salmonella paradigm.

The recent emergence of food-borne pathogens, such as Salmonella enterica serotype Enteritidis (S. enteritidis) and Escherichia coli O157:H7, has generated increasing interest in how infectious diseases can invade, persist and spread within new host populations. To alter their host range pathogens require adaptations, which ensure their circulation in a new animal population. Adaptations for circulation in different populations of vertebrate hosts seem to have been acquired multiple times within the genus Salmonella because extant Salmonella serotypes differ greatly with regard to host range. In this article, mechanisms involved in host adaptation are deduced by considering the influence of the host immune response on circulation of Salmonella serotypes within populations of vertebrate animals. This approach contributes to the identification of genes involved in host adaptation and provides new insights into the emergence of food-borne pathogens.

SspA is required for lethal Salmonella enterica serovar Typhimurium infections in calves but is not essential for diarrhea.

Salmonella pathogenicity island 1 (SPI-1) encodes virulence determinants, which are important for enteropathogenicity in calves. To determine whether the Salmonella enterica serovar Typhimurium SPI-1 effector proteins SspA and SptP are important for enteropathogenicity, strains lacking these proteins were tested during oral infection of calves. Calves infected with a sptP mutant or its isogenic parent developed diarrhea and lethal morbidity. In contrast, calves infected with an sspA mutant developed diarrhea, which resolved within 10 days but did not result in mortality. The sspA mutant was recovered from bovine intestinal tissues at numbers similar to those obtained for its isogenic parent and caused marked intestinal lesions. Thus, the severity of pathological changes caused by serovar Typhimurium strains or their ability to cause diarrhea were not predictive of their ability to cause lethal morbidity in calves. We conclude that factors other than or in addition to bacterial colonization, intestinal lesions, or electrolyte loss contribute to lethal morbidity in calves infected with serovar Typhimurium.

The shdA gene is restricted to serotypes of Salmonella enterica subspecies I and contributes to efficient and prolonged fecal shedding.

Little is known about factors which enable Salmonella serotypes to circulate within populations of livestock and domestic fowl. We have identified a DNA region which is present in Salmonella serotypes commonly isolated from livestock and domestic fowl (S. enterica subspecies I) but absent from reptile-associated Salmonella serotypes (S. bongori and S. enterica subspecies II to VII). This DNA region was cloned from Salmonella serotype Typhimurium and sequence analysis revealed the presence of a 6,105-bp open reading frame, designated shdA, whose product's deduced amino acid sequence displayed homology to that of AIDA-I from diarrheagenic Escherichia coli, MisL of serotype Typhimurium, and IcsA of Shigella flexneri. The shdA gene was located adjacent to xseA at 52 min, in a 30-kb DNA region which is not present in Escherichia coli K-12. A serotype Typhimurium shdA mutant was shed with the feces in reduced numbers and for a shorter period of time compared to its isogenic parent. A possible role for the shdA gene during the expansion in host range of S. enterica subspecies I to include warm-blooded vertebrates is discussed.

Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems.

Salmonellae encode two virulence-associated type III secretion systems (TTSS) within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Two Salmonella typhimurium genes, sspH1 and sspH2, that encode proteins similar to the Shigella flexneri and Yersinia species TTSS substrates, IpaH and YopM, were identified. SspH1 and SspH2 are proteins containing leucine-rich repeats that are differentially targeted to the SPI1 and SPI2 TTSS. sspH2 transcription was induced within RAW264.7 macrophages, and was dependent upon the SPI2-encoded regulator ssrA/ssrB. In contrast, sspH1 transcription is independent of SPI2, and is not induced after bacterial phagocytosis by eukaryotic cells. Infection of eukaryotic cells with strains expressing a SspH2-CyaA fusion protein resulted in SPI2 TTSS-dependent cAMP increases. In contrast, SspH1-CyaA-mediated cAMP increases were both SPI1 and SPI2 TTSS dependent. sspH2-like sequences were found in most Salmonella serotypes examined, whereas sspH1 was detected in only one S. typhimurium isolate, indicating that the copy number of sspH genes can be variable within Salmonella serotypes. S. typhimurium deleted for both sspH1 and sspH2 was not able to cause a lethal infection in calves, indicating that these genes participate in S. typhimurium virulence for animals.

Ferrioxamine-mediated Iron(III) utilization by Salmonella enterica.

Utilization of ferrioxamines as sole sources of iron distinguishes Salmonella enterica serotypes Typhimurium and Enteritidis from a number of related species, including Escherichia coli. Ferrioxamine supplements have therefore been used in preenrichment and selection media to increase the bacterial growth rate while selectivity is maintained. We characterized the determinants involved in utilization of ferrioxamines B, E, and G by S. enterica serotype Typhimurium by performing siderophore cross-feeding bioassays. Transport of all three ferric siderophores across the outer membrane was dependent on the FoxA receptor encoded by the Fur-repressible foxA gene. However, only the transport of ferrioxamine G was dependent on the energy-transducing protein TonB, since growth stimulation of a tonB strain by ferrioxamines B and E was observed, albeit at lower efficiencies than in the parental strain. Transport across the inner membrane was dependent on the periplasmic binding protein-dependent ABC transporter complex comprising FhuBCD, as has been reported for other hydroxamate siderophores of enteric bacteria. The distribution of the foxA gene in the genus Salmonella, as indicated by DNA hybridization studies and correlated with the ability to utilize ferrioxamine E, was restricted to subspecies I, II, and IIIb, and this gene was absent from subspecies IIIa, IV, VI, and VII (formerly subspecies IV) and Salmonella bongori (formerly subspecies V). S. enterica serotype Typhimurium mutants with either a transposon insertion or a defined nonpolar frameshift (+2) mutation in the foxA gene were not able to utilize any of the three ferrioxamines tested. A strain carrying the nonpolar foxA mutation exhibited a significantly reduced ability to colonize rabbit ileal loops compared to the foxA+ parent. In addition, a foxA mutant was markedly attenuated in mice inoculated by either the intragastric or intravenous route. Mice inoculated with the foxA mutant were protected against subsequent challenge by the foxA+ parent strain.

Of mice, calves, and men. Comparison of the mouse typhoid model with other Salmonella infections.

Numerous Salmonella typhimurium virulence factors have been identified and characterized using experimental infection of mice. While the murine typhoid model has been used successfully for Salmonella typhi vaccine development and to infer virulence mechanisms important during typhoid fever, information derived from infection of mice has been of limited value in elucidating the mechanisms by which S. typhimurium causes enteritis in humans. Progress in our understanding of virulence mechanisms contributing to diarrheal disease comes from recent studies of bovine enteritis, a S. typhimurium infection, which manifests as acute gastroenteritis. This review compares virulence genes and mechanisms required during murine typhoid, typhoid fever, and bovine enteritis. Comparison of illnesses caused in different animal hosts identifies virulence mechanisms involved in species specific disease manifestations. The determination of the relative importance of virulence factors for disease manifestations in different host species provides an important link between the in vitro characterization of genes and their role during host pathogen interaction.

Expression and transcriptional control of the Salmonella typhimurium Ipf fimbrial operon by phase variation.

The lpf operon mediates adhesion of Salmonella typhimurium to murine Peyer's patches. To investigate expression of this virulence factor, a transcriptional fusion between the lpf operon and the genes lacZYA of Escherichia coli was constructed and introduced into the S. typhimurium chromosome. The resulting strain yielded both Lac+ and Lac- colony phenotypes. Alternation between Lac+ (phase ON) and Lac- (phase OFF) phenotypes occurred by a heritable phase variation mechanism, as inoculation of broth cultures with bacteria picked from a Lac+ colony gave rise to a considerably higher proportion of Lac+ colonies than inoculation with bacteria picked from a Lac- colony. During growth in vitro, phase transition from ON to OFF and from OFF to ON occurred at rates 6.8 x 10(-3) and 2.4 x 10(-4) events per cell per generation respectively. In a murine intestinal organ culture model, selection for the ON expression state occurred when attached bacteria were recovered from Peyer's patches, suggesting that Lac phase variation correlated with expression of lpf mediated adherence. Selection for either the ON or the OFF expression state of the Ipf operon in vivo was studied in mice immunized with either GST or GST-LpfA fusion protein. A strong selection against phase ON cells occurred only in animals immunized with GST-LpfA. The ability of animals immunized with GST-LpfA to distinguish between phase ON and phase OFF bacteria provided evidence for the presence of LpfA fimbrial protein in phase ON cells and for its exposure to the immune system.