ResearchPad - klebsiella https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Within-patient plasmid dynamics in <i>Klebsiella pneumoniae</i> during an outbreak of a carbapenemase-producing <i>Klebsiella pneumoniae</i>]]> https://www.researchpad.co/article/elastic_article_15752 Knowledge of within-patient dynamics of resistance plasmids during outbreaks is important for understanding the persistence and transmission of plasmid-mediated antimicrobial resistance. During an outbreak of a Klebsiella pneumoniae carbapenemase-producing (KPC) K. pneumoniae, the plasmid and chromosomal dynamics of K. pneumoniae within-patients were investigated.MethodsDuring the outbreak, all K. pneumoniae isolates of colonized or infected patients were collected, regardless of their susceptibility pattern. A selection of isolates was short-read and long-read sequenced. A hybrid assembly of the short-and long-read sequence data was performed. Plasmid contigs were extracted from the hybrid assembly, annotated, and within patient plasmid comparisons were performed.ResultsFifteen K. pneumoniae isolates of six patients were short-read whole-genome sequenced. Whole-genome multi-locus sequence typing revealed a maximum of 4 allele differences between the sequenced isolates. Within patients 1 and 2 the resistance gene- and plasmid replicon-content did differ between the isolates sequenced. Long-read sequencing and hybrid assembly of 4 isolates revealed loss of the entire KPC-gene containing plasmid in the isolates of patient 2 and a recombination event between the plasmids in the isolates of patient 1. This resulted in two different KPC-gene containing plasmids being simultaneously present during the outbreak.ConclusionDuring a hospital outbreak of a KPC-producing K. pneumoniae isolate, plasmid loss of the KPC-gene carrying plasmid and plasmid recombination was detected within the isolates from two patients. When investigating outbreaks, one should be aware that plasmid transmission can occur and the possibility of within- and between-patient plasmid variation needs to be considered. ]]> <![CDATA[A prospective study of bloodstream infections among febrile adolescents and adults attending Yangon General Hospital, Yangon, Myanmar]]> https://www.researchpad.co/article/elastic_article_13833 Bloodstream infection (BSI) is common among persons seeking healthcare for severe febrile illness in low-and middle-income countries. Data on community-onset BSI are few for some countries in Asia, including Myanmar. Such data are needed to inform empiric antimicrobial treatment of patients and to monitor and control antimicrobial resistance. We performed a one year, prospective study collecting information and blood cultures from patients presenting with fever at a tertiary referral hospital in Yangon, Myanmar. We found that almost 10% of participants had a bloodstream infection, and that Salmonella enterica serovars Typhi and Paratyphi A were the most common pathogens. Typhoidal Salmonella were universally resistant to ciprofloxacin. More than half of Escherichia coli and Klebsiella pneumoniae were resistant to extended-spectrum cephalosporins and resistance to carbapenems was also identified in some isolates. We show that typhoid and paratyphoid fever are common, and fluoroquinolone resistance is widespread. Extended-spectrum cephalosporin resistance is common in E. coli and K. pneumoniae and carbapenem resistance is present. Our findings inform empiric antimicrobial management of severe febrile illness, underscore the value of routine use of blood cultures, indicate that measures to prevent and control enteric fever are warranted, and suggest a need to monitor and mitigate antimicrobial resistance among community-acquired pathogens.

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<![CDATA[Neonatal sepsis in Iran: A systematic review and meta-analysis on national prevalence and causative pathogens]]> https://www.researchpad.co/article/Nc80eb6d8-6c1e-4acb-a5fa-a329abafdd28

Background

Neonatal sepsis is accounted for 30–50% of annual neonatal deaths in developing countries. We performed a systematic review and meta-analysis study to evaluate the national prevalence and identification of the etiological pathogens of neonatal sepsis in Iran.

Methods

A comprehensive literature search was done on the national and international databases for studies published between 2000 and 2019. The DerSimonian and Laird random-effects model was used to calculate pooled prevalence estimates, with 95% confidence intervals (CIs). Subgroup analyses and meta-regressions regarding the gender, type of sepsis and time during were also performed. Data were extracted, analyzed, and presented according to PRISMA guideline.

Results

Of 944 publications identified, 22 studies containing 14,683 neonates met the eligibility criteria. The pooled national prevalence of sepsis in Iran was 15.98% (95%CI, 11.96–20.46%; 1,367/14,683). Prevalence rate in boys (20.42%; 95%CI, 9.03–34.8%) was slightly higher than girls (18.5%; 95%CI, 7.4–32.8). A decreasing trend in prevalence of neonatal sepsis was found in recent years, although not statistically significant (c = -0.005; P value = 0.4). The most prevalent causative bacterial pathogens were Enterobacter spp. (23.04%), followed by Klebsiella pneumoniae (17.54%), coagulase-negative Staphylococci (14.06%), Escherichia coli (13.92%), Pseudomonas aeruginosa (12.67%), and Staphylococcus aureus (11.48%).

Conclusion

Our findings showed a high prevalence of neonatal sepsis in suspected neonates, suggesting the need to implement preventive measures, routine assessment, and close monitoring of neonates. Also, Enterobacter spp. and Klebsiella pneumoniae were identified as the principal bacterial pathogens responsible for neonatal septicemia in Iran.

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<![CDATA[The successful containment of a hospital outbreak caused by NDM-1-producing Klebsiella pneumoniae ST307 using active surveillance]]> https://www.researchpad.co/article/5c6dc9abd5eed0c484529fbf

The worldwide dissemination of high-risk carbapenemase-producing Klebsiella pneumoniae clones has become a major threat to healthcare facilities. This study describes the successful containment of a hospital outbreak caused by NDM-1-producing K. pneumoniae Sequence Type (ST) 307 using active surveillance. The outbreak began when a patient was transferred from a local hospital. After 48 hours in our hospital, a tracheal aspirate was positive for a meropenem resistant and carbapenemase-producing K. pneumoniae. All patients in the medical intensive care unit (ICU) and the neurology wards were subject to contact precautions. The hospital surfaces and devices, healthcare workers, and patients from these wards were screened by cultures. Fecal swabs were placed into broth and PCR for blaKPC, blaOXA-48, blaIMP, blaVIM, and blaNDM, which were performed directly from the broth after 12 hours. PCRs were also performed on DNA extracted from carbapenemase-producing species from subcultured broths. Five and nine days later, two more patients’ rectal swabs tested positive. Molecular assays identified K. pneumoniae blaNDM-1 onto a 130-kb conjugative plasmid (IncY, IncFIIs, and IncFIIY), ST307. After the three patients were discharged, monitoring continued, and after three weeks with negative results, rectal swabbing ended. In conclusion, it was possible to contain a hospital outbreak caused by NDM-1-producing K. pneumoniae ST307 through epidemiological and microbiological surveillance. With the methodology used, the detection of NDM-type genes in fecal samples was obtained in approximately 15 hours after obtaining the fecal sample.

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<![CDATA[Accelerated bacterial detection in blood culture by enhanced acoustic flow cytometry (AFC) following peptide nucleic acid fluorescence in situ hybridization (PNA-FISH)]]> https://www.researchpad.co/article/5c6730b6d5eed0c484f37f63

Bacteraemia is a risk factor for subsequent clinical deterioration and death. Current reliance on culture-based methods for detection of bacteraemia delays identification and assessment of this risk until after the optimal period for positively impacting treatment decisions has passed. Therefore, a method for rapid detection and identification of bacterial infection in the peripheral bloodstream in acutely ill patients is crucial for improved patient survival through earlier targeted antibiotic treatment. The turnaround time for current clinical laboratory methods ranges from 12 to 48 hours, emphasizing the need for a faster diagnostic test. Here we describe a novel assay for accelerated generic detection of bacteria in blood culture (BC) using peptide nucleic acid fluorescence in situ hybridization enhanced acoustic flow cytometry (PNA-FISH-AFC). For assay development, we used simulated blood cultures (BCs) spiked with one of three bacterial species at a low starting concentration of 10 CFU/mL: Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa. Under current clinical settings, it takes a minimum of 12 hours incubation to reach positivity on the BacTEC system, corresponding to a bacterial concentration of 107−109 CFU/mL optimal for further analyses. In contrast, our PNA-FISH-AFC assay detected 103–104 CFU/mL bacteria in BC following a much shorter culture incubation of 5 to 10 hours. Using either PCR-based FilmArray assay or MALDI-TOF for bacterial detection, it took 7–10 and 12–24 hours of incubation, respectively, to reach the positive result. These findings indicate a potential time advantage of PNA-FISH-AFC assay for rapid bacterial detection in BC with significantly improved turnaround time over currently used laboratory techniques.

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<![CDATA[Bridging antimicrobial resistance knowledge gaps: The East African perspective on a global problem]]> https://www.researchpad.co/article/5c6b2668d5eed0c484289a21

Background

There is worldwide concern of rapidly increasing antimicrobial resistance (AMR). However, there is paucity of resistance surveillance data and updated antibiograms in Africa in general. This study was undertaken in Kenyatta National Hospital (KNH) -the largest public tertiary referral centre in East & Central Africa—to help bridge existing AMR knowledge and practice gaps.

Methods

A retrospective review of VITEK 2 (bioMérieux) records capturing antimicrobial susceptibility data for the year 2015 was done and analysed using WHONET and SPSS.

Results

Analysis of 624 isolates revealed AMR rates higher than most recent local and international reports. 88% of isolates tested were multi-drug resistant (MDR) whereas 26% were extensively-drug resistant (XDR). E. coli and K. pneumoniae had poor susceptibility to penicillins (8–48%), cephalosporins (16–43%), monobactams (17–29%), fluoroquinolones (22–44%) and trimethoprim-sulfamethoxazole (7%). Pseudomonas aeruginosa and Acinetobacter baumanii were resistant to penicillins and cephalosporins, with reduced susceptibility to carbapenems (70% and 27% respectively). S aureus had poor susceptibility to penicillins (3%) and trimethoprim-sulfamethoxazole (29%) but showed excellent susceptibility to imipenem (90%), vancomycin (97%) and linezolid (99%).

Conclusions

The overwhelming resistance to commonly used antibiotics heralds a clarion call towards strengthening antimicrobial stewardship programmes and regular AMR regional surveillance.

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<![CDATA[Flow cytometry, a powerful novel tool to rapidly assess bacterial viability in metal working fluids: Proof-of-principle]]> https://www.researchpad.co/article/5c5df33bd5eed0c484580f75

Metalworking fluids (MWF) are water- or oil-based liquids to cool and lubricate tools, work pieces and machines, inhibit corrosion and remove swarf. One of the major problems in the MWF industry is bacterial growth as bacterial enzymes can cause MWF degradation. In addition, bacteria can form biofilms which hamper the functioning of machines. Last but not least, some bacterial by-products are toxic (e.g. endotoxins) and present potential health risks for metalworking machine operators via the formation of aerosols. Therefore, a novel fast yet accurate analytical method to evaluate and predict the antibacterial capacity of MWF would be an important asset. As such a tool is currently lacking, the present study aimed to develop a protocol based on flow cytometry (FCM) to assess the antibacterial potential of newly developed MWF independent of bacterial growth. Results of this novel method were compared to a biochallenge test currently used in MWF industry and also to traditional plate counts. Our results represent a proof-of-principle that FCM can reliably predict the antibacterial capacity of MWF already within one day of incubation with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis, being substantially faster than the current growth-based methods.

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<![CDATA[Nontherapeutic equivalence of a generic product of imipenem-cilastatin is caused more by chemical instability of the active pharmaceutical ingredient (imipenem) than by its substandard amount of cilastatin]]> https://www.researchpad.co/article/5c648cbfd5eed0c484c81696

Background

We demonstrated therapeutic nonequivalence of “bioequivalent” generics for meropenem, but there is no data with generics of other carbapenems.

Methods

One generic product of imipenem-cilastatin was compared with the innovator in terms of in vitro susceptibility testing, pharmaceutical equivalence, pharmacokinetic (PK) and pharmacodynamic (PD) equivalence in the neutropenic mouse thigh, lung and brain infection models. Both pharmaceutical forms were then subjected to analytical chemistry assays (LC/MS).

Results and conclusion

The generic product had 30% lower concentration of cilastatin compared with the innovator of imipenem-cilastatin. Regarding the active pharmaceutical ingredient (imipenem), we found no differences in MIC, MBC, concentration or potency or AUC, confirming equivalence in terms of in vitro activity. However, the generic failed therapeutic equivalence in all three animal models. Its Emax against S. aureus in the thigh model was consistently lower, killing from 0.1 to 7.3 million less microorganisms per gram in 24 hours than the innovator (P = 0.003). Against K. pneumoniae in the lung model, the generic exhibited a conspicuous Eagle effect fitting a Gaussian equation instead of the expected sigmoid curve of the Hill model. In the brain infection model with P. aeruginosa, the generic failed when bacterial growth was >4 log10 CFU/g in 24 hours, but not if it was less than 2.5 log10 CFU/g. These large differences in the PD profile cannot be explained by the lower concentration of cilastatin, and rather suggested a failure attributable to the imipenem constituent of the generic product. Analytical chemistry assays confirmed that, besides having 30% less cilastatin, the generic imipenem was more acidic, less stable, and exhibited four different degradation masses that were absent in the innovator.

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<![CDATA[ESBL colonization and acquisition in a hospital population: The molecular epidemiology and transmission of resistance genes]]> https://www.researchpad.co/article/5c466564d5eed0c484518ef0

A prospective cohort study (German Clinical Trial Registry, No. 00005273) was performed to determine pre-admission colonization rates, hospital acquisition risk factors, subsequent infection rates and colonization persistence including the respective molecular epidemiology and transmission rates of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (EPE). A total of 342 EPEs were isolated from rectal swabs of 1,334 patients on admission, at discharge and 6 months after hospitalization. Inclusion criteria were patients’ age > 18 years, expected length of stays > 48 hours, external referral. The EPEs were characterized by routine microbiological methods, a DNA microarray and ERIC-PCR. EPE colonization was found in 12.7 % of admitted patients, with the highest rate (23.8 %) in patients from nursing homes. During hospitalization, 8.1 % of the patients were de novo EPE colonized, and invasive procedures, antibiotic and antacid therapies were independent risk factors. Only 1/169 patients colonized on admission developed a hospital-acquired EPE infection. Escherichia coli was the predominant EPE (88.9 %), and 92.1% of the ESBL phenotypes could be related to CTX-M variants with CTX-M-1/15 group being most frequent (88.9%). A corresponding β-lactamase could not be identified in five isolates. Hospital-acquired EPE infections in patients colonized before or during hospitalization were rare. The diversity of the EPE strains was much higher than that of the underlying plasmids. In seven patients, transmission of the respective plasmid across different species could be observed indicating that the current strain-based surveillance approaches may underestimate the risk of inter-species transmission of resistance genes.

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<![CDATA[Correlation of Klebsiella pneumoniae Comparative Genetic Analyses with Virulence Profiles in a Murine Respiratory Disease Model]]> https://www.researchpad.co/article/5989da28ab0ee8fa60b817cb

Klebsiella pneumoniae is a bacterial pathogen of worldwide importance and a significant contributor to multiple disease presentations associated with both nosocomial and community acquired disease. ATCC 43816 is a well-studied K. pneumoniae strain which is capable of causing an acute respiratory disease in surrogate animal models. In this study, we performed sequencing of the ATCC 43816 genome to support future efforts characterizing genetic elements required for disease. Furthermore, we performed comparative genetic analyses to the previously sequenced genomes from NTUH-K2044 and MGH 78578 to gain an understanding of the conservation of known virulence determinants amongst the three strains. We found that ATCC 43816 and NTUH-K2044 both possess the known virulence determinant for yersiniabactin, as well as a Type 4 secretion system (T4SS), CRISPR system, and an acetonin catabolism locus, all absent from MGH 78578. While both NTUH-K2044 and MGH 78578 are clinical isolates, little is known about the disease potential of these strains in cell culture and animal models. Thus, we also performed functional analyses in the murine macrophage cell lines RAW264.7 and J774A.1 and found that MGH 78578 (K52 serotype) was internalized at higher levels than ATCC 43816 (K2) and NTUH-K2044 (K1), consistent with previous characterization of the antiphagocytic properties of K1 and K2 serotype capsules. We also examined the three K. pneumoniae strains in a novel BALB/c respiratory disease model and found that ATCC 43816 and NTUH-K2044 are highly virulent (LD50<100 CFU) while MGH 78578 is relatively avirulent.

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<![CDATA[Differences in Inflammatory Response Induced by Two Representatives of Clades of the Pandemic ST258 Klebsiella pneumoniae Clonal Lineage Producing KPC-Type Carbapenemases]]> https://www.researchpad.co/article/5989daeeab0ee8fa60bc060f

ST258-K. pneumoniae (ST258-KP) strains, the most widespread multidrug-resistant hospital-acquired pathogens, belong to at least two clades differing in a 215 Kb genomic region that includes the cluster of capsule genes. To investigate the effects of the different capsular phenotype on host-pathogen interactions, we studied representatives of ST258-KP clades, KKBO-1 and KK207-1, for their ability to activate monocytes and myeloid dendritic cells from human immune competent hosts. The two ST258-KP strains strongly induced the production of inflammatory cytokines. Significant differences between the strains were found in their ability to induce the production of IL-1β: KK207-1/clade I was much less effective than KKBO-1/clade II in inducing IL-1β production by monocytes and dendritic cells. The activation of NLRP3 inflammasome pathway by live cells and/or purified capsular polysaccharides was studied in monocytes and dendritic cells. We found that glibenclamide, a NLRP3 inhibitor, inhibits more than 90% of the production of mature IL-1β induced by KKBO1 and KK207-1. KK207-1 was always less efficient compared to KKBO-1 in: a) inducing NLRP3 and pro-IL-1β gene and protein expression; b) in inducing caspase-1 activation and pro-IL-1β cleavage. Capsular composition may play a role in the differential inflammatory response induced by the ST258-KP strains since capsular polysaccharides purified from bacterial cells affect NLRP3 and pro-IL-1β gene expression through p38MAPK- and NF-κB-mediated pathways. In each of these functions, capsular polysaccharides from KK207-1 were significantly less efficient compared to those purified from KKBO-1. On the whole, our data suggest that the change in capsular phenotype may help bacterial cells of clade I to partially escape innate immune recognition and IL-1β-mediated inflammation.

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<![CDATA[Step-Wise Increase in Tigecycline Resistance in Klebsiella pneumoniae Associated with Mutations in ramR, lon and rpsJ]]> https://www.researchpad.co/article/5989d9e8ab0ee8fa60b6bf4d

Klebsiella pneumoniae is a gram-negative bacterium that causes numerous diseases, including pneumonia and urinary tract infections. An increase in multidrug resistance has complicated the treatment of these bacterial infections, and although tigecycline shows activity against a broad spectrum of bacteria, resistant strains have emerged. In this study, the whole genomes of two clinical and six laboratory-evolved strains were sequenced to identify putative mutations related to tigecycline resistance. Of seven tigecycline-resistant strains, seven (100%) had ramR mutations, five (71.4%) had lon mutations, one (14.2%) had a ramA mutation, and one (14.2%) had an rpsJ mutation. A higher fitness cost was observed in the laboratory-evolved strains but not in the clinical strains. A transcriptome analysis demonstrated high expression of the ramR operon and acrA in all tigecycline-resistant strains. Genes involved in nitrogen metabolism were induced in the laboratory-evolved strains compared with the wild-type and clinical strains, and this difference in nitrogen metabolism reflected the variation between the laboratory-evolved and the clinical strains. Complementation experiments showed that both the wild-type ramR and the lon genes could partially restore the tigecycline sensitivity of K. pneumoniae. We believe that this manuscript describes the first construct of a lon mutant in K. pneumoniae, which allowed confirmation of its association with tigecycline resistance. Our findings illustrate the importance of the ramR operon and the lon and rpsJ genes in K. pneumoniae resistance to tigecycline.

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<![CDATA[High Prevalence of New Delhi Metallo-β-Lactamase-1 (NDM-1) Producers among Carbapenem-Resistant Enterobacteriaceae in Kuwait]]> https://www.researchpad.co/article/5989da90ab0ee8fa60b9fe99

The aim of the study was to determine the prevalence of New Delhi metallo-β lactamase-1 (NDM-1) producing Enterobacteriaceae in Kuwait over a one year period. Consecutive Enterobacteriaceae isolates with reduced susceptibility to carbapenems were collected from four government hospitals in Kuwait from January–December 2014. Their susceptibility to 18 antibiotics was performed by determining the minimum inhibitory concentration. Isolates resistant to carbapenems were tested by PCR for resistant genes. Finger printing of the positive isolates was done by DiversiLab®. Clinical data of patients harboring NDM-1 positive isolates were analyzed. A total of 764 clinically significant Enterobacteriaceae isolates were studied. Of these, 61 (8%) were carbapenem-resistant. Twenty one out of these 61 (34.4%) were NDM-1-producers. All patients positive for NDM-1-carrying bacteria were hospitalized. About half were females (11/21 [52.3%]), average age was 53.3 years and the majority were Kuwaitis (14/21 [66.6%]). Six patients (28.5%) gave a history of travel or healthcare contact in an endemic area. Mortality rate was relatively high (28.6%). The predominant organism was Klebsiella pneumoniae (14 [66.6%]) followed by E. coli (4 [19%]). All NDM-1-positive isolates were resistant to meropenem, ertapenem, cefotaxime, cefoxitin and ampicillin, while 95.2% were resistant to imipenem, cefepime, and piperacillin-tazobactam. They were multidrug resistant including resistance to tigecycline, but 90% remained susceptible to colistin. About two-thirds of isolates (61.9%) co-produced-extended spectrum β-lactamases. During the study period, an outbreak of NDM-1 positive K. pneumoniae occurred in one hospital involving 3 patients confirmed by DiversiLab® analysis. In conclusion, NDM-1-producing Enterobacteriaceae is a growing healthcare problem with increasing prevalence in Kuwait, especially in hospitalized patients, leaving few therapeutic options. A high prevalence of NDM-1 necessitates the implementation of strict infection control to prevent the spread of these organisms.

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<![CDATA[Non-Traditional Antibacterial Screening Approaches for the Identification of Novel Inhibitors of the Glyoxylate Shunt in Gram-Negative Pathogens]]> https://www.researchpad.co/article/5989db15ab0ee8fa60bccfcc

Antibacterial compounds that affect bacterial viability have traditionally been identified, confirmed, and characterized in standard laboratory media. The historical success of identifying new antibiotics via this route has justifiably established a traditional means of screening for new antimicrobials. The emergence of multi-drug-resistant (MDR) bacterial pathogens has expedited the need for new antibiotics, though many in the industry have questioned the source(s) of these new compounds. As many pharmaceutical companies' chemical libraries have been exhaustively screened via the traditional route, we have concluded that all compounds with any antibacterial potential have been identified. While new compound libraries and platforms are being pursued, it also seems prudent to screen the libraries we currently have in hand using alternative screening approaches. One strategy involves screening under conditions that better reflect the environment pathogens experience during an infection, and identifying in vivo essential targets and pathways that are dispensable for growth in standard laboratory media in vitro. Here we describe a novel screening strategy for identifying compounds that inhibit the glyoxylate shunt in Pseudomonas aeruginosa, a pathway that is required for bacterial survival in the pulmonary environment. We demonstrate that these compounds, which were not previously identified using traditional screening approaches, have broad-spectrum antibacterial activity when they are tested under in vivo-relevant conditions. We also show that these compounds have potent activity on both enzymes that comprise the glyoxylate shunt, a feature that was supported by computational homology modeling. By dual-targeting both enzymes in this pathway, we would expect to see a reduced propensity for resistance development to these compounds. Taken together, these data suggest that understanding the in vivo environment that bacterial pathogens must tolerate, and adjusting the antibacterial screening paradigm to reflect those conditions, could identify novel antibiotics for the treatment of serious MDR pathogens.

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<![CDATA[Evaluation of a modified meropenem hydrolysis assay on a large cohort of KPC and VIM carbapenemase-producing Enterobacteriaceae]]> https://www.researchpad.co/article/5989db51ab0ee8fa60bdc3dd

Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. The introduction of rapid and sensitive methods for the detection of carbapenemase-producing bacteria is of increasing importance. The carbapenemase production can be detected using non-molecular methods (such as the modified Hodge test, the synergy test, the Carba NP test and the antibiotic hydrolysis assays) and DNA-based methods. In this study, we propose a modified version of a previously described meropenem hydrolysis assay (MHA) by MALDI-TOF MS for the phenotypic detection in 2h of carbapenemase-producing Enterobacteriaceae. The MHA was successfully applied to detect carbapenemase activity in 981 well-characterized Enterobacteriaceae strains producing KPC or VIM carbapenemases, and in 146 carbapenem fully susceptible strains. This assay, applied also to NDM and OXA-48-producing strains and to CRE with resistance mechanisms other than carbapenemase production, has proved to be able to distinguish between carbapenemase-producing and -nonproducing Enterobacteriaceae.

As already stated and as observed in our hands, MHA by MALDI-TOF MS analysis is independent from the type of carbapenemases involved, it is faster and easier to perform/interpret than culture-based methods. On the other hand, it cannot detect other carbapenem resistance mechanisms, such as porin alterations and efflux mechanisms.

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<![CDATA[SCOTTI: Efficient Reconstruction of Transmission within Outbreaks with the Structured Coalescent]]> https://www.researchpad.co/article/5989da56ab0ee8fa60b8eef1

Exploiting pathogen genomes to reconstruct transmission represents a powerful tool in the fight against infectious disease. However, their interpretation rests on a number of simplifying assumptions that regularly ignore important complexities of real data, in particular within-host evolution and non-sampled patients. Here we propose a new approach to transmission inference called SCOTTI (Structured COalescent Transmission Tree Inference). This method is based on a statistical framework that models each host as a distinct population, and transmissions between hosts as migration events. Our computationally efficient implementation of this model enables the inference of host-to-host transmission while accommodating within-host evolution and non-sampled hosts. SCOTTI is distributed as an open source package for the phylogenetic software BEAST2. We show that SCOTTI can generally infer transmission events even in the presence of considerable within-host variation, can account for the uncertainty associated with the possible presence of non-sampled hosts, and can efficiently use data from multiple samples of the same host, although there is some reduction in accuracy when samples are collected very close to the infection time. We illustrate the features of our approach by investigating transmission from genetic and epidemiological data in a Foot and Mouth Disease Virus (FMDV) veterinary outbreak in England and a Klebsiella pneumoniae outbreak in a Nepali neonatal unit. Transmission histories inferred with SCOTTI will be important in devising effective measures to prevent and halt transmission.

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<![CDATA[Multi-scale model of drug induced adaptive resistance of Gram-negative bacteria to polymyxin B]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc0ff

The purpose of this report is to apply multi-scale modeling using the theory of physiologically structured populations (PSP) to develop a mathematical model for antimicrobial resistance based on a heterogeneous distribution of receptors and affinities among bacterial cells. The theory has been tested on data obtained from an in vitro static time-kill infection model analyzing the pharmacodynamics of polymyxin B against Gram-negative bacteria. The drug binding parameter KD (dissociation equilibrium constant) is assumed to vary between the bacterial cells. The PSP model describes the time course of the density distribution of KD upon exposure to cytotoxic drug concentrations. The drug increases the hazard of cell death as a function of receptor occupancy. The initial distribution of KD is described by the Weibull function. Time-kill data were used for model qualification. In vitro static time-kill experiments to evaluate the rate and extent of killing due to polymyxin B against two Klebsiella pneumoniae clinical isolates with differing susceptibilities to polymyxin B were performed over 48 h. The time-kill kinetics data of bacterial load cfu (colony forming units)/mL was used for model qualification. The resistant bacterial population is determined by the balance between growth rate and hazard of cell death controlled by polymyxin B concentrations. There exists a critical KD value below which cells continue to grow. Estimates of shape parameters for distributions of KD yielded unimodal distributions with the modes at 0 nM and the right tails containing approximately 25% of the bacteria. Our findings support a hypothesis that resistance of Klebsiella pneumoniae to polymyxin B can be at least partially attributed to a drug-induced selection of a subpopulation due to heterogeneity of polymyxin B receptor binding in the bacterial population.

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<![CDATA[Prevalence of MDR pathogens of bacterial meningitis in Egypt and new synergistic antibiotic combinations]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc5b1

The aim of this study was identifying bacterial pathogens involved in meningitis, studying their antibiotic resistance profiles, investigating the antibiotic resistance genes as well as evaluating the use of various antibiotic combinations. Antibiotic susceptibility tests were evaluated according to CLSI guidelines. Antibiotic combinations were evaluated by calculating the Fractional Inhibitory Concentration (FIC) index. A total of 71 bacterial isolates were recovered from 68 culture positive CSF specimens. Sixty five of these isolates (91.5%) were recovered from single infection specimens, while 6 isolates (8.4%) were recovered from mixed infection specimens. Out of the 71 recovered isolates, 48 (67.6%) were Gram-positive, and 23 (32.4%) were Gram-negative. Thirty one of the Gram positive isolates were S. pneumoniae (64.6%, n = 48). Out of the recovered 71 isolates; 26 (36.6%) were multidrug-resistant (MDR) isolates of which, 18 (69.2%) were Gram-negative and 8 (30.8%) were Gram-positive. All MDR isolates (100%) showed resistance to penicillin and ampicillin, however, they showed lower resistance to meropenem (50%), levofloxacin (50%), amikacin (48%), pipercillin-tazobactam (45.8%). Most common antibiotic resistance genes were investigated including: tem (21.1%), shv (15.8%), ctx-m (15.8%) coding for TEM-, SHV, CTX-M extended-spectrum beta-lactamases (ESBLs), respectively; aac(6')-I b(26.3%) coding for aminoglycoside 6’-N-acetyltransferase type Ib ciprofloxacin resistant variant; and qnrA (5.3%) gene coding for quinolone resistance. The DNA sequences of the respective resistance genes of some selected isolates were PCR amplified, analyzed and submitted to the GenBank database under the accession numbers, KX214665, KX214664, KX214663, KX214662, respectively. The FIC values for ampicillin/sulbactam plus cefepime showed either additive or synergistic effect against ten tested Gram-negative MDR isolates, while doxycycline plus levofloxacin combination revealed synergism against two MDR Gram-positive isolates. The results indicate high prevalence of antibiotic resistance among MDR isolates. Therefore, new guidelines should be implemented in Egypt to rationalize the use and avoid the misuse and abuse of antimicrobial agents.

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<![CDATA[Antibodies Directed against a Peptide Epitope of a Klebsiella pneumoniae-Derived Protein Are Present in Ankylosing Spondylitis]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdb875

Ankylosing spondylitis (AS) is a chronic inflammatory arthritis of unknown origin. Its autoimmune origin has been suggested but never proven. Several reports have implicated Klebsiella pneumoniae as a triggering or perpetuating factor in AS; however, its role in the disease pathogenesis remains debated. Moreover, despite extensive investigations, a biomarker for AS has not yet been identified. To clarify these issues, we screened a random peptide library with pooled IgGs obtained from 40 patients with AS. A peptide (AS peptide) selected from the library was recognized by serum IgGs from 170 of 200 (85%) patients with AS but not by serum specimens from 100 healthy controls. Interestingly, the AS peptide shows a sequence similarity with several molecules expressed at the fibrocartilaginous sites that are primarily involved in the AS inflammatory process. Moreover, the peptide is highly homologous to a Klebsiella pneumoniae dipeptidase (DPP) protein. The antibody affinity purified against the AS peptide recognizes the autoantigens and the DPP protein. Furthermore, serum IgG antibodies against the Klebsiella DPP121-145 peptide epitope were detected in 190 of 200 patients with AS (95%), 3 of 200 patients with rheumatoid arthritis (1.5%) and only 1 of 100 (1%) patients with psoriatic arthritis. Such reactivity was not detected in healthy control donors. Our results show that antibodies directed against an epitope of a Klebsiella pneumoniae-derived protein are present in nearly all patients with AS. In the absence of serological biomarkers for AS, such antibodies may represent a useful tool in the diagnosis of the disease.

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<![CDATA[Process Optimization on Micro-Aeration Supply for High Production Yield of 2,3-Butanediol from Maltodextrin by Metabolically-Engineered Klebsiella oxytoca]]> https://www.researchpad.co/article/5989dafdab0ee8fa60bc563a

An optimization process with a cheap and abundant substrate is considered one of the factors affecting the price of the production of economical 2,3-Butanediol (2,3-BD). A combination of the conventional method and response surface methodology (RSM) was applied in this study. The optimized levels of pH, aeration rate, agitation speed, and substrate concentration (maltodextrin) were investigated to determine the cost-effectiveness of fermentative 2,3-BD production by metabolically-engineered Klebsiella oxytoca KMS005. Results revealed that pH, aeration rate, agitation speed, and maltodextrin concentration at levels of 6.0, 0.8 vvm, 400 rpm, and 150 g/L respectively were the optimal conditions. RSM also indicated that the agitation speed was the most influential parameter when either agitation and aeration interaction or agitation and substrate concentration interaction played important roles for 2,3-BD production by the strain from maltodextrin. Under interim fed-batch fermentation, 2,3-BD concentration, yield, and productivity were obtained at 88.1±0.2 g/L, 0.412±0.001 g/g, and 1.13±0.01 g/L/h respectively within 78 h.

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