Host adaptation and convergent evolution increases antibiotic resistance without loss of virulence in a major human pathogen.

As human population density and antibiotic exposure increase, specialised bacterial subtypes have begun to emerge. Arising among species that are common commensals and infrequent pathogens, antibiotic-resistant 'high-risk clones' have evolved to better survive in the modern human. Here, we show that the major matrix porin (OmpK35) of Klebsiella pneumoniae is not required in the mammalian host for colonisation, pathogenesis, nor for antibiotic resistance, and that it is commonly absent in pathogenic isolates. This is found in association with, but apparently independent of, a highly specific change in the co-regulated partner porin, the osmoporin (OmpK36), which provides enhanced antibiotic resistance without significant loss of fitness in the mammalian host. These features are common in well-described 'high-risk clones' of K. pneumoniae, as well as in unrelated members of this species and similar adaptations are found in other members of the Enterobacteriaceae that share this lifestyle. Available sequence data indicate evolutionary convergence, with implications for the spread of lethal antibiotic-resistant pathogens in humans.

Longitudinal estimation of Plasmodium falciparum prevalence in relation to malaria prevention measures in six sub-Saharan African countries.

BACKGROUND: Plasmodium falciparum prevalence (PfPR) is a widely used metric for assessing malaria transmission intensity. This study was carried out concurrently with the RTS,S/AS01 candidate malaria vaccine Phase III trial and estimated PfPR over ≤ 4 standardized cross-sectional surveys. METHODS: This epidemiology study (NCT01190202) was conducted in 8 sites from 6 countries (Burkina Faso, Gabon, Ghana, Kenya, Malawi, and Tanzania), between March 2011 and December 2013. Participants were enrolled in a 2:1:1 ratio according to age category: 6 months-4 years, 5-19 years, and ≥ 20 years, respectively, per year and per centre. All sites carried out surveys 1-3 while survey 4 was conducted only in 3 sites. Surveys were usually performed during the peak malaria parasite transmission season, in one home visit, when medical history and malaria risk factors/prevention measures were collected, and a blood sample taken for rapid diagnostic test, microscopy, and haemoglobin measurement. PfPR was estimated by site and age category. RESULTS: Overall, 6401 (survey 1), 6411 (survey 2), 6400 (survey 3), and 2399 (survey 4) individuals were included in the analyses. In the 6 months-4 years age group, the lowest prevalence (assessed using microscopy) was observed in 2 Tanzanian centres (4.6% for Korogwe and 9.95% for Bagamoyo) and Lambaréné, Gabon (6.0%), while the highest PfPR was recorded for Nanoro, Burkina Faso (52.5%). PfPR significantly decreased over the 3 years in Agogo (Ghana), Kombewa (Kenya), Lilongwe (Malawi), and Bagamoyo (Tanzania), and a trend for increased PfPR was observed over the 4 surveys for Kintampo, Ghana. Over the 4 surveys, for all sites, PfPR was predominantly higher in the 5-19 years group than in the other age categories. Occurrence of fever and anaemia was associated with high P. falciparum parasitaemia. Univariate analyses showed a significant association of anti-malarial treatment in 4 surveys (odds ratios [ORs]: 0.52, 0.52, 0.68, 0.41) and bed net use in 2 surveys (ORs: 0.63, 0.68, 1.03, 1.78) with lower risk of malaria infection. CONCLUSION: Local PfPR differed substantially between sites and age groups. In children 6 months-4 years old, a significant decrease in prevalence over the 3 years was observed in 4 out of the 8 study sites. Trial registration Clinical Trials.gov identifier: NCT01190202:NCT. GSK Study ID numbers: 114001.

Predictability of Phenotype in Relation to Common ß-Lactam Resistance Mechanisms in Escherichia coli and Klebsiella pneumoniae.

The minimal concentration of antibiotic required to inhibit the growth of different isolates of a given species with no acquired resistance mechanisms has a normal distribution. We have previously shown that the presence or absence of transmissible antibiotic resistance genes has excellent predictive power for phenotype. In this study, we analyzed the distribution of six ß-lactam antibiotic susceptibility phenotypes associated with commonly acquired resistance genes in Enterobacteriaceae in Sydney, Australia. Escherichia coli (n = 200) and Klebsiella pneumoniae (n = 178) clinical isolates, with relevant transmissible resistance genes (blaTEM, n = 33; plasmid AmpC, n = 69; extended-spectrum ß-lactamase [ESBL], n = 116; and carbapenemase, n = 100), were characterized. A group of 60 isolates with no phenotypic resistance to any antibiotics tested and carrying none of the important ß-lactamase genes served as comparators. The MICs for all drug-bacterium combinations had a normal distribution, varying only in the presence of additional genes relevant to the phenotype or, for ertapenem resistance in K. pneumoniae, with a loss or change in the outer membrane porin protein OmpK36. We demonstrated mutations in ompK36 or absence of OmpK36 in all isolates in which reduced susceptibility to ertapenem (MIC, >1 mg/liter) was evident. Ertapenem nonsusceptibility in K. pneumoniae was most common in the context of an OmpK36 variant with an ESBL or AmpC gene. Surveillance strategies to define appropriate antimicrobial therapies should include genotype-phenotype relationships for all major transmissible resistance genes and the characterization of mutations in relevant porins in organisms, like K. pneumoniae.

Phenotypic detection of carbapenemase-producing Enterobacteriaceae by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry and the Carba NP test.

We compared the diagnostic accuracy of the Carba NP test with that of a straightforward matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method for detecting carbapenemase-producing Enterobacteriaceae (CPE). Using PCR as the reference method, both tests demonstrated a sensitivity of 87% and a specificity of 100%. MALDI-TOF MS offers a potential alternative for the rapid detection of CPE in the clinical laboratory setting.

Comparison of the novel Partec rapid malaria test to the conventional Giemsa stain and the gold standard real-time PCR.

Malaria remains the single most frequent cause of death in Africa, killing one child every 30 s, but treatment decisions are often made only on clinical diagnosis, as laboratory techniques to confirm the clinical suspicion are labor intensive and costly. In this study, we evaluated the recently developed Partec rapid malaria test (PM) for the detection of Plasmodium spp. in human blood from patients in an area where malaria is endemic and compared the results with those of thick blood film Giemsa stain (GS) in terms of its performance and operational characteristics, using real-time (RT) PCR as the gold standard. The sensitivities of the PM and the GS were 62.2% (95% CI, 56.3 to 67.8) and 61.8% (95% CI, 55.9 to 67.4), respectively, while the specificities were 96.0% (95% CI, 92.3 to 98.3) and 98% (95% CI, 95.0 to 99.5), respectively. There was an excellent agreement between the results for the PM and those of the GS (k [level of agreement] = 0.96; P < 0.001). The results for the PM were obtained more quickly and at less cost than those for the GS. The performance characteristics of the PM were almost equal to those of the GS, but the operational characteristics were better, and the PM can therefore be considered as an alternative method for GS.

Systemic bacteraemia in children presenting with clinical pneumonia and the impact of non-typhoid salmonella (NTS).

BACKGROUND: The diagnosis and antimicrobial treatment of pneumonia in African children in the absence of diagnostic means such as x-ray facilities or microbiological laboratories relies primarily on clinical symptoms presented by the patients. In order to assess the spectrum of bacterial pathogens, blood cultures were performed in children fulfilling the clinical criteria of pneumonia. METHODS: In total, 1032 blood cultures were taken from children between 2 months and 5 years of age who were admitted to a rural hospital in Ghana between September 2007 and July 2009. Pneumonia was diagnosed clinically and according to WHO criteria classified as "non-severe pneumonia" and "severe pneumonia" ("severe pneumonia" includes the WHO categories "severe pneumonia" and "very severe pneumonia"). RESULTS: The proportion of bacteriaemia with non-typhoid salmonella (NTS) was similar in children with pneumonia (16/173, 9.2%) compared to children hospitalized for other reasons (112/859, 13%). NTS were the predominant organisms isolated from children with clinical pneumonia and significantly more frequent than Streptococcus pneumoniae (8/173, 4.6%). Nine percent (9/101) of children presenting with severe pneumonia and 10% (7/72) of children with non-severe pneumonia were infected with NTS. Nineteen out of 123 NTS isolates (15%) were susceptible to aminopenicillins (amoxycillin/ampicillin), 23/127 (18%) to chlorampenicol, and 23/98 (23%) to co-trimoxazole. All NTS isolates were sensitive to ceftriaxone and ciprofloxacin. CONCLUSION: In Sub-saharan Africa, sepsis with NTS should be considered in children with symptoms of pneumonia and aminopenicillins might often not be the adequate drugs for treatment.

blaCTX-M-15 carried by IncF-type plasmids is the dominant ESBL gene in Escherichia coli and Klebsiella pneumoniae at a hospital in Ghana.

Escherichia coli and Klebsiella pneumoniae producing extended-spectrum ß-lactamases (ESBLs) are among the most multidrug-resistant pathogens in hospitals and are spreading worldwide. Horizontal gene transfer and spread of high-risk clones are involved in ESBL dissemination. Investigation of the resistance phenotypes of 101 consecutive clinical E. coli (n=58) and K. pneumoniae (n=43) isolated at the Komfo Anokye Teaching Hospital in Ghana over 3 months revealed 63 (62%) with an ESBL phenotype. All 63 had a blaCTX-M gene, and sequence analysis showed that 62 of these were blaCTX-M-15. blaCTX-M-15 was linked to ISEcp1 and orf477Δ in all isolates, and most isolates also carried blaTEM, aac(3)-II, aacA4cr, and/or blaOXA-30 genes on IncF plasmids. XbaI/pulsed-field electrophoresis showed heterogeneity among isolates of both species, suggesting that blaCTX-M-15 dissemination is caused by horizontal gene transfer rather than clonal spread of these species in Ghana.

Clinical indicators for bacterial co-infection in Ghanaian children with P. falciparum infection.

Differentiation of infectious causes in severely ill children is essential but challenging in sub- Saharan Africa. The aim of the study was to determine clinical indicators that are able to identify bacterial co-infections in P. falciparum infected children in rural Ghana. In total, 1,915 severely ill children below the age of 15 years were recruited at Agogo Presbyterian Hospital in Ghana between May 2007 and February 2011. In 771 (40%) of the children malaria parasites were detected. This group was analyzed for indicators of bacterial co-infections using bivariate and multivariate regression analyses with 24 socio-economic variables, 16 terms describing medical history and anthropometrical information and 68 variables describing clinical symptoms. The variables were tested for sensitivity, specificity, positive predictive value and negative predictive value. In 46 (6.0%) of the children with malaria infection, bacterial co-infection was detected. The most frequent pathogens were non-typhoid salmonellae (45.7%), followed by Streptococcus spp. (13.0%). Coughing, dehydration, splenomegaly, severe anemia and leukocytosis were positively associated with bacteremia. Domestic hygiene and exclusive breastfeeding is negatively associated with bacteremia. In cases of high parasitemia (>10,000/µl), a significant association with bacteremia was found for splenomegaly (OR 8.8; CI 1.6-48.9), dehydration (OR 18.2; CI 2.0-166.0) and coughing (OR 9.0; CI 0.7-118.6). In children with low parasitemia, associations with bacteremia were found for vomiting (OR 4.7; CI 1.4-15.8), severe anemia (OR 3.3; CI 1.0-11.1) and leukocytosis (OR 6.8 CI 1.9-24.2). Clinical signs of impaired microcirculation were negatively associated with bacteremia. Ceftriaxone achieved best coverage of isolated pathogens. The results demonstrate the limitation of clinical symptoms to determine bacterial co-infections in P. falciparum infected children. Best clinical indicators are dependent on the parasitemia level. Even with a moderate sensitivity of >60%, only low positive predictive values can be obtained due to low prevalence of bacteremia. Rapid testing for distinguishing parasitemia and bacteremia is essential.

Incidence and characteristics of bacteremia among children in rural Ghana.

The objective of the study was to describe systemic bacterial infections occurring in acutely ill and hospitalized children in a rural region in Ghana, regarding frequency, incidence, antimicrobial susceptibility patterns and associations with anthropometrical data.Blood cultures were performed in all children below the age of five years, who were admitted to Agogo Presbyterian Hospital (APH), Asante Region, Ghana, between September 2007 and July 2009. Medical history and anthropometrical data were assessed using a standardized questionnaire at admission. Incidences were calculated after considering the coverage population adjusted for village-dependent health-seeking behavior.Among 1,196 hospitalized children, 19.9% (n = 238) were blood culture positive. The four most frequent isolated pathogens were nontyphoidal salmonellae (NTS) (53.3%; n = 129), Staphylococcus aureus (13.2%; n = 32), Streptococcus pneumoniae (9.1%; n = 22) and Salmonella ser. Typhi (7.0%; n = 17). Yearly cumulative incidence of bacteremia was 46.6 cases/1,000 (CI 40.9-52.2). Yearly cumulative incidences per 1,000 of the four most frequent isolates were 25.2 (CI 21.1-29.4) for NTS, 6.3 (CI 4.1-8.4) for S. aureus, 4.3 (CI 2.5-6.1) for S. pneumoniae and 3.3 (CI 1.8-4.9) for Salmonella ser. Typhi. Wasting was positively associated with bacteremia and systemic NTS bloodstream infection. Children older than three months had more often NTS bacteremia than younger children. Ninety-eight percent of NTS and 100% of Salmonella ser. Typhi isolates were susceptible to ciprofloxacin, whereas both tested 100% susceptible to ceftriaxone. Seventy-seven percent of NTS and 65% of Salmonella ser. Typhi isolates were multi-drug resistant (MDR). Systemic bacterial infections in nearly 20% of hospitalized children underline the need for microbiological diagnostics, to guide targeted antimicrobial treatment and prevention of bacteremia. If microbiological diagnostics are lacking, calculated antimicrobial treatment of severely ill children in malaria-endemic areas should be considered.