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BackgroundCulture-based microbiological investigation of hospital-acquired or ventilator-associated pneumonia (HAP or VAP) is insensitive, with aetiological agents often unidentified. This can lead to excess antimicrobial treatment of patients with susceptible pathogens, while those with resistant bacteria are treated inadequately for prolonged periods. Using PCR to seek pathogens and their resistance genes directly from clinical samples may improve therapy and stewardship.MethodsSurplus routine lower respiratory tract samples were collected from intensive care unit patients about to receive new or changed antibiotics for hospital-onset lower respiratory tract infections at 15 UK hospitals. Testing was performed using the BioFire FilmArray Pneumonia Panel (bioMérieux) and Unyvero Pneumonia Panel (Curetis). Concordance analysis compared machine and routine microbiology results, while Bayesian latent class (BLC) analysis estimated the sensitivity and specificity of each test, incorporating information from both PCR panels and routine microbiology.FindingsIn 652 eligible samples; PCR identified pathogens in considerably more samples compared with routine microbiology: 60.4% and 74.2% for Unyvero and FilmArray respectively vs 44.2% by routine microbiology. PCR tests also detected more pathogens per sample than routine microbiology. For common HAP/VAP pathogens, FilmArray had sensitivity of 91.7%–100.0% and specificity of 87.5%–99.5%; Unyvero had sensitivity of 50.0%–100.0%%, and specificity of 89.4%–99.0%. BLC analysis indicated that, compared with PCR, routine microbiology had low sensitivity, ranging from 27.0% to 69.4%.InterpretationConventional and BLC analysis demonstrated that both platforms performed similarly and were considerably more sensitive than routine microbiology, detecting potential pathogens in patient samples reported as culture negative. The increased sensitivity of detection realised by PCR offers potential for improved antimicrobial prescribing.

BackgroundAntibiotics are extensively prescribed in intensive care units (ICUs), yet little is known about how antibiotic-related decisions are made in this setting. We explored how beliefs, perceptions and contextual factors influenced ICU clinicians’ antibiotic prescribing.MethodsWe conducted 4 focus groups and 34 semistructured interviews with clinicians involved in antibiotic prescribing in four English ICUs. Focus groups explored factors influencing prescribing, whereas interviews examined decision-making processes using two clinical vignettes. Data were analysed using thematic analysis, applying the Necessity Concerns Framework.ResultsClinicians’ antibiotic decisions were influenced by their judgement of the necessity for prescribing/not prescribing, relative to their concerns about potential adverse consequences. Antibiotic necessity perceptions were strongly influenced by beliefs that antibiotics would protect patients from deterioration and themselves from the ethical and legal consequences of undertreatment. Clinicians also reported concerns about prescribing antibiotics. These generally centred on antimicrobial resistance; however, protecting the individual patient was prioritised over these societal concerns. Few participants identified antibiotic toxicity concerns as a key influencer. Clinical uncertainty often complicated balancing antibiotic necessity against concerns. Decisions to start or continue antibiotics often represented ‘erring on the side of caution’ as a protective response in uncertainty. This approach was reinforced by previous experiences of negative consequences (‘being burnt’) which motivated prescribing ‘just in case’ of an infection. Prescribing decisions were also context-dependent, exemplified by a lower perceived threshold to prescribe antibiotics out-of-hours, input from external team members and local prescribing norms.ConclusionEfforts to improve antibiotic stewardship should consider clinicians’ desire to protect with a prescription. Rapid molecular microbiology, with appropriate communication, may diminish clinicians’ fears of not prescribing or of using narrower-spectrum antibiotics.

We agree with Che et al. (1) that understanding how mobile genetic elements (MGEs) spread antimicrobial resistance (AMR) is important. However, decades of research have already characterized a diverse toolbox of MGEs involved in the emergence of AMR, including conjugative plasmids and insertion sequences (ISs), and their interactions. “Mobile” AMR generally arises following rare capture of a chromosomal gene by a particular “intracellularly mobile” MGE, e.g., an IS or a transposon (Tn), and translocation to “intercellularly mobile” MGEs [plasmids, phage, integrative elements (2, 3)]. Tools for systematic analysis of AMR gene−MGE interactions in mounting sequence data are needed, but incorporating existing biological knowledge into their design is vital, as are representative benchmarking datasets and recognition of data biases and resulting limitations. Che et al. screened bacterial plasmid sequences for markers to classify them as nonmobilizable (no relaxase), mobilizable (relaxase), or conjugative (relaxase+). Relying on the absence of a predicted feature is risky, and Plascad’s poor sensitivity in many species was not recognized, due to a taxonomically inadequate benchmark dataset. Numerous conjugative plasmids, and close relatives, from low-GC gram-positive organisms (dataset S1 in ref. 1; e.g., Staphylococcus, pSK41, pWBG4, pWBG749; Clostridium, pCW3, pCP13; Enterococcus/Streptococcus , pAMβ1, pCF10, pRE25, pMG1, pIP501) (3⇓–5 … [↵][1]1To whom correspondence may be addressed. Email: sally.partridge{at}health.nsw.gov.au or neville.firth{at}sydney.edu.au. [1]: #xref-corresp-1-1

Background Rapid molecular diagnostic tests to investigate the microbial aetiology of pneumonias may improve treatment and antimicrobial stewardship in intensive care units (ICUs). Clinicians’ endorsement and uptake of these tests is crucial to maximise engagement; however, adoption may be impeded if users harbour unaddressed concerns or if device usage is incompatible with local practice. Accordingly, we strove to identify ICU clinicians’ beliefs about molecular diagnostic tests for pneumonias before implementation at the point-of-care. Methods We conducted semi-structured interviews with 35 critical care doctors working in four ICUs in the United Kingdom. A clinical vignette depicting a fictitious patient with signs of pneumonia was used to explore clinicians’ beliefs about the importance of molecular diagnostics and their concerns. Data were analysed thematically. Results Clinicians’ beliefs about molecular tests could be grouped into two categories: perceived potential of molecular diagnostics to improve antibiotic prescribing (Molecular Diagnostic Necessity) and concerns about how the test results could be implemented into practice (Molecular Diagnostic Concerns). Molecular Diagnostic Necessity stemmed from beliefs that positive results would facilitate targeted antimicrobial therapy; that negative results would signal the absence of a pathogen, and consequently that having the molecular diagnostic results would bolster clinicians’ prescribing confidence. Molecular Diagnostic Concerns included unfamiliarity with the device’s capabilities, worry that it would detect non-pathogenic bacteria, uncertainty whether it would fail to detect pathogens, and discomfort with withholding antibiotics until receiving molecular test results. Conclusions Clinicians believed rapid molecular diagnostics for pneumonias were potentially important and were open to using them; however, they harboured concerns about the tests’ capabilities and integration into clinical practice. Implementation strategies should bolster users’ necessity beliefs while reducing their concerns; this can be accomplished by publicising the tests’ purpose and benefits, identifying and addressing clinicians’ misconceptions, establishing a trial period for first-hand familiarisation, and emphasising that, with a swift (e.g., 60–90 min) test, antibiotics can be started and refined after molecular diagnostic results become available.

SummaryBackgroundICU patients with hospital-acquired or ventilator-associated pneumonia (HAP or VAP) have high mortality, so broad-spectrum antibiotics are initiated at clinical diagnosis, then refined after 2-3 days, once microbiology results become available. Unfortunately, culture-based microbiological investigation is also insensitive, with aetiological agents remaining unidentified in many cases. This leads to extended over-treatment of patients with susceptible pathogens, whilst those with highly-resistant pathogens are treated inadequately for prolonged periods. Using PCR to seek pathogens and their resistance genes directly from clinical samples may improve therapy and stewardship. The INHALE study compared two PCR platforms for HAP/VAP diagnosis against routine microbiology (RM), identifying one to progress into a Randomised Controlled Trial (RCT).MethodsSurplus routine sputa, endotracheal tube exudates and bronchoalveolar lavages were collected from ICU patients about to receive new or changed antibiotics for hospital-onset lower respiratory tract infections at 15 UK hospitals. Samples were tested (or frozen for testing) within 72h of collection. Testing was performed using the BioFire FilmArray Pneumonia Panel (bioMérieux) and Unyvero Pneumonia Panel (Curetis). Agreement between machine- and RM-results was categorised as ‘full positive/negative concordance’, ‘partial concordance’ or ‘major/minor discordance’. Bayesian latent class (BLC) analysis was used to estimate the sensitivity and specificity of each test, incorporating information from both PCR panels, 16S rDNA analysis and RM.FindingsIn 652 eligible samples; PCR identified pathogens in considerably more samples compared with RM: 60.4% and 74.2% for Unyvero and FilmArray respectively vs. 44.2%. Both tests also recorded more organisms per sample than routine culture, with the two PCR tests frequently in agreement with each other. For common HAP/VAP pathogens, FilmArray had sensitivity of 91.7-100.0% and specificity of 87.5-99.5%; Unyvero had sensitivity of 83.3-100.0%% except for Klebsiella aerogenes (50.0%) and Serratia marcescens (77.8%), and specificity of 89.4-99.0%. BLC analysis indicated that, compared with PCR, RM had low sensitivity, ranging from 27.0% to 69.4% for common respiratory pathogens. PCR detected more high-consequence antimicrobial resistance genes than would have been predicted by RM and susceptibility testing; around half the host strains could be detected when culture was repeated and they were sought assiduously.InterpretationConventional and BLC analysis demonstrated that both platforms performed similarly and were considerably more sensitive than RM, detecting potential pathogens in patient samples reported as culture negative. FilmArray had slightly higher sensitivity than Unyvero for common pathogens and was chosen for INHALE’s RCT, based on the balance of these results, a swifter turnaround time (75 min vs. 6h), and a smaller footprint. The increased sensitivity of detection realised by PCR offers potential for improved antimicrobial prescribing.

IntroductionSeveral viral respiratory infections - notably influenza - are associated with secondary bacterial infection and additional pathology. The extent to which this applies for COVID-19 is unknown. Accordingly, we aimed to define the bacteria causing secondary pneumonias in COVID-19 ICU patients using the FilmArray Pneumonia Panel, and to determine this test’s potential in COVID-19 management.MethodsCOVID-19 ICU patients with clinically-suspected secondary infection at 5 UK hospitals were tested with the FilmArray at point of care. We collected patient demographic data and compared FilmArray results with routine culture.ResultsWe report results of 110 FilmArray tests on 94 patients (16 had 2 tests): 69 patients (73%) were male, the median age was 59 yrs; 92 were ventilated. Median hospital stay before testing was 14 days (range 1-38). Fifty-nine (54%) tests were positive, with 141 bacteria detected. Most were Enterobacterales (n=55, includingKlebsiellaspp. [n= 35]) orStaphylococcus aureus(n=13), as is typical of hospital and ventilator pneumonia. Community pathogens, includingHaemophilus influenzae(n=8) andStreptococcus pneumoniae(n=1), were rarer. FilmArray detected one additional virus (Rhinovirus/Enterovirus) and no atypical bacteria. Fewer samples (28 % vs. 54%) were positive by routine culture, and fewer species were reported per sample;Klebsiellaspecies remained the most prevalent pathogens.ConclusionFilmArray had a higher diagnostic yield than culture for ICU COVID-19 patients with suspected secondary pneumonias. The bacteria found mostly were Enterobacterales,S. aureusandP. aeruginosa, as in typical HAP/VAP, but withKlebsiellaspp. more prominent. We found almost no viral co-infection. Turnaround from sample to results is around 1h 15 min compared with the usual 72h for culture, giving prescribers earlier data to inform antimicrobial decisions.

This is a narrative review on the potential of rapid and point-of-care microbiological testing in pneumonia patients, focusing particularly on hospital-acquired and ventilator-associated pneumonia, which have substantial mortality and diverse microbiology. This work is written from a United Kingdom perspective, but much of it is generalizable internationally. In a world where antimicrobial resistance is a major international threat, the use of rapid molecular diagnostics has great potential to improve both the management of pneumonia patients and the stewardship of antibiotics. Rapid tests potentially can distinguish patients with bacterial versus viral infection and can swiftly identify bacterial pathogens and their resistances. We seek to answer the question: "Can such tests be used as an antibiotic guardian?" Their availability at the bedside rather than in the laboratory should best ensure that results are swiftly used to optimize patient management but will raise new challenges, not the least with respect to maintaining quality control and microbiology/infection control input. A further challenge lies in assessing the degree of trust that treating clinicians will place in these molecular diagnostic tests, particularly when early de-escalation of antibiotic therapy is indicated.

Summary Background Hospital-acquired pneumonia (HAP) is pneumonia that occurs ≥48 h after hospital admission; it is the most common hospital-acquired infection contributing to death. Ventilator-associated pneumonia (VAP) arises ≥48–72 h after intubation. Opinions differ on whether VAP is a subset of HAP; the same pathogens predominate in both. Compared with VAP-free controls, patients developing VAP are twice as likely to die and have significantly longer stays in intensive care units. Guidelines recommend that microbiological cultures should guide antibiotic treatment, but these lack sensitivity and take 48–72 h to process, meaning that initial therapy must be empiric, generally with broad-spectrum agents. Given increasing pressure to improve both antibiotic stewardship and patient outcomes, the National Institute for Health and Care Excellence and the Infectious Diseases Society of America recommend research into rapid molecular diagnostic tests to identify causative organisms and their antibiotic resistances. Ideally, these would supersede culture, being quicker and more sensitive. In the UK, the INHALE research programme, funded by the National Institute for Health Research, is exploring rapid molecular diagnostics to inform treatment of HAP/VAP and, given resource implications, incorporates a health economic component. Aim To identify previous economic modelling of HAP/VAP costs to inform this component. Methods Literature review of HAP/VAP studies with economic modelling identified from three databases. Findings Twenty studies were identified. Only one study specifically evaluated strategies to improve diagnosis; the remaining 19 studies omitted this important aspect. Conclusion HAP/VAP modelling would be improved by better awareness of long-term outcomes and treatment complexity. To the authors' knowledge, no similar literature reviews of economic modelling for HAP/VAP have been published.

Background: O’Neill’s recent Review on Antimicrobial Resistance expressed the view that by 2020 high-income countries should make it mandatory to support antimicrobial prescribing with rapid diagnostic evidence whenever possible. Methods: Routine microbiology diagnosis of 95 respiratory specimens from patients with severe infection were compared with those generated by the Unyvero P55 test, which detects 20 pathogens and 19 antimicrobial resistance markers. Supplementary molecular testing for antimicrobial resistance genes, comprehensive culture methodology and 16S rRNA sequencing were performed. Results: Unyvero P55 produced 85 valid results, 67% of which were concordant with those from the routine laboratory. Unyvero P55 identified more potential pathogens per specimen than routine culture (1.34 vs. 0.47 per specimen). Independent verification using 16S rRNA sequencing and culture (n = 10) corroborated 58% of additional detections compared to routine microbiology. Overall the average sensitivity for organism detection by Unyvero P55 was 88.8% and specificity was 94.9%. While Unyvero P55 detected more antimicrobial resistance markers than routine culture, some instances of phenotypic resistance were missed. Conclusions: The Unyvero P55 is a rapid pathogen detection test for lower respiratory specimens, which identifies a larger number of pathogens than routine microbiology. The clinical significance of these additional organisms is yet to be determined. Further studies are required to determine the effect of the test in practise on antimicrobial prescribing and patient outcomes. Keywords: Pneumonia, PCR, Diagnostics, Antimicrobial resistance, 16S metagenomics, Rapid testing

Background: In sub-Saharan Africa, there is scanty data on the causes of neonatal sepsis and antimicrobial resistance among common invasive pathogens that might guide policy and practice. Methods: A cross-sectional observational prevalence and etiology study of neonates with suspected sepsis admitted to the neonatal intensive care unit, University Teaching Hospital, Lusaka, Zambia, between October 2013 and May 2014. Data from blood cultures and phenotypic antibiotic susceptibility testing were compared with multivariate analysis of risk factors for neonatal sepsis. Results: Of 313 neonates with suspected sepsis, 54% (170/313) were male; 20% (62/313) were born to HIV-positive mothers; 33% (103/313) had positive blood cultures, of which 85% (88/103) were early-onset sepsis. Klebsiella species was the most prevalent isolate, accounting for 75% (77/103) of cases, followed by coagulase-negative staphylococci 6% (7/103)], Staphylococcus aureus 6% (6/103)], Escherichia coli 5% (5/103)] and Candida species 5% (5/103)]. For Klebsiella species, antibiotic resistance ranged from 96%-99% for World Health Organizationrecommended first-line therapy (gentamicin and ampicillin/penicillin) to 94%-97% for third-generation cephalosporins. The prevalence of cultureconfirmed sepsis increased from 0 to 39% during the period December 2013 to March 2014, during which time mortality increased 29%-47%; 93% (14/15) of late-onset sepsis and 82% (37/45) of early-onset sepsis aged 4-7 days were admitted > 2 days before the onset of symptoms. Culture results for only 25% (26/103) of cases were available before discharge or death. Maternal HIV infection was associated with a reduced risk of neonatal sepsis odds ratio, 0.46 (0.23-0.93); P = 0.029]. Conclusions: Outbreaks of nosocomial multiantibiotic-resistant infections are an important cause of neonatal sepsis and associated mortality. Reduced risk of neonatal sepsis associated with maternal HIV infection is counterintuitive and requires further investigation.

During an investigation of CRISPR carriage in clinical, multi-drug resistant, Klebsiella pneumoniae, a novel CRISPR-Cas system (which we have designated Type IV-B) was detected on plasmids from two K. pneumoniae isolates from Egypt (isolated in 2002-2003) and a single K. pneumoniae isolate from the UK (isolated in 2017). Sequence analysis of other genomes available in GenBank revealed that this novel Type IV-B CRISPR-Cas system was present on 28 other plasmids from various Enterobacteriaceae hosts and was never found on the chromosome. Type IV-B is found exclusively on IncHI1B/IncFIB plasmids and is associated with multiple putative transposable elements. Type IV-B has a single repeat-spacer array (CRISPR1) upstream of the cas loci with some spacers matching regions of conjugal transfer genes of IncFIIK/IncFIB(K) plasmids suggesting a role in plasmid incompatibility. Expression of the cas loci was confirmed in available clinical isolates by RT-PCR; indicating the system is active. To our knowledge, this is the first report describing a new subtype within Type IV CRISPR-Cas systems exclusively associated with IncHI1B/IncFIB plasmids.ImportanceHere, we report the identification of a novel subtype of Type IV CRISPR-Cas that is expressed and exclusively carried by IncHI1B/IncFIB plasmids in Enterobacteriaceae, demonstrating unique evolutionarily juxtaposed connections between CRISPR-Cas and mobile genetic elements (MGEs). Type IV-B encodes a variety of spacers showing homology to DNA from various sources, including plasmid specific spacers and is therefore thought to provide specific immunity against plasmids of other incompatible groups (IncFIIK/IncFIB(K)). The relationship between Type IV-B CRISPR-Cas and MGEs that surround and interrupt the system is likely to promote rearrangement and be responsible for the observed variability of this type. Finally, the Type IV-B CRISPR-Cas is likely to co-operate with other cas loci within the bacterial host genome during spacer acquisition.

Summary Respiratory tract infections rank second as causes of adult and paediatric morbidity and mortality worldwide. Respiratory tract infections are caused by many different bacteria (including mycobacteria) and viruses, and rapid detection of pathogens in individual cases is crucial in achieving the best clinical management, public health surveillance, and control outcomes. Further challenges in improving management outcomes for respiratory tract infections exist: rapid identification of drug resistant pathogens; more widespread surveillance of infections, locally and internationally; and global responses to infections with pandemic potential. Developments in genome amplification have led to the discovery of several new respiratory pathogens, and sensitive PCR methods for the diagnostic work-up of these are available. Advances in technology have allowed for development of single and multiplexed PCR techniques that provide rapid detection of respiratory viruses in clinical specimens. Microarray-based multiplexing and nucleic-acid-based deep-sequencing methods allow simultaneous detection of pathogen nucleic acid and multiple antibiotic resistance, providing further hope in revolutionising rapid point of care respiratory tract infection diagnostics.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated cas genes are sequence-specific DNA nuclease systems found in bacteria and archaea. CRISPR/Cas systems use RNA transcripts of previously acquired DNA (spacers) to target invading genetic elements with the same sequence, including plasmids. In this research we studied the relationship between CRISPR/Cas systems and multidrug resistance in Escherichia coli . The presence of Type I-E and Type I-F CRISPR systems was investigated among 82 antimicrobial-susceptible and 96 MDR clinical E. coli isolates by PCR and DNA sequencing. Phylogrouping and MLST were performed to determine relatedness of isolates. RT-PCR was performed to ascertain the expression of associated cas genes. Type I-F CRISPR was associated with the B2 phylogroup and was significantly overrepresented in the susceptible group (22.0%) compared with the MDR group (2.1%). The majority of CRISPR I-F-containing isolates had spacer sequences that matched IncF and IncI plasmids. RT-PCR demonstrated that Type I-F cas genes were expressed and therefore potentially functional. The CRISPR I-F system is more likely to be found in antimicrobial-susceptible E. coli . Given that the Type I-F system is expressed in WT isolates, we suggest that this difference could be due to the CRISPR system potentially interfering with the acquisition of antimicrobial resistance plasmids, maintaining susceptibility in these isolates.

This review, for the occasion of the 40th anniversary of the Journal of Antimicrobial Chemotherapy (JAC), gives an overview of the manuscripts related to veterinary bacteriology published in the journal in the past 40 years with a focus on ‘One Health’ aspects. From 1975 to 2000 the number of manuscripts related to veterinary medicine was limited, but thereafter, the number steadily increased. Most manuscripts published were related to food-producing animals, but companion animals and minor species were also covered. Subjects included antimicrobial usage in animals and the consequences for human medicine, new resistance genes and mechanisms, the prevalence and epidemiology of antimicrobial resistance, and the emergence of resistant bacteria in animals with zoonotic potential such as livestock-associated MRSA (LA-MRSA), methicillin-resistant Staphylococcus pseudintermedius (MRSP) and ESBL-producing Enterobacteriaceae. These manuscripts have added to our knowledge on the risks of transmission of resistant bacteria from animals to humans and the importance of the prudent use of antimicrobial agents in veterinary medicine.

This study aimed to evaluate the performance of the Unyvero P50 pneumonia assay, the first ‘sample-in, answer-out’ system for rapid identification of pathogens and antibiotic resistance markers directly from clinical specimens. Overall, Unyvero P50 displayed very good sensitivity (>95%); however, specificity was low (33%) mainly because 40% of the specimens were reported as normal flora. Specifically, one or more pathogens were identified in 28 of them. From a detailed analysis of 42 specimens selected at random, 76% of the additionally reported pathogens were confirmed present in primary specimens. Detection of selected resistance markers was compared to routine phenotypic susceptibility testing, supplemented with Checkpoints microarray system, PCR and sequencing. Concordance was mixed, primarily due to issues with panel’s choice of markers and detection of some intrinsic beta-lactamases. Finally, we offer a critical analysis of the assay’s microbial panel and resistance markers and provide suggestions for improvement.

The strategy of decreasing antimicrobial prescribing to reduce existing antimicrobial resistance appears attractive, but its effectiveness, particularly in the community setting, remains unclear. Contrasting results obtained from the relatively few studies in this area confuse matters further. Prescribing reductions have successfully reduced the prevalence of resistance among respiratory pathogens, but in these cases single bacterial clones dominated the resistant population. In contrast, this strategy has not succeeded in reducing plasmid-encoded resistance among Escherichia coli. The reasons why some prescribing restriction policies are more successful than others are complex, with the three key determinants being the fitness cost of resistance, the clonal structure of the resistant bacterial population and co-selection of resistant organisms by other antimicrobials. The resistant bacterial phenotypes that are likely to be the easiest to eliminate will be those composed of relatively clonal populations that bear a fitness cost of resistance and are not significantly subjected to co-selection by other antimicrobials. Plasmid-encoded resistance seldom meets these criteria and, hence, is likely to be the most difficult to reduce.

The mechanochemical synthesis and characterization of a zinc complex with famotidine is described. The complex was characterized by microanalysis and a number of spectroscopic techniques. The complex was of M:L dihydrate type. Derivatization of famotidine with zinc appears to enhance the activity of the drug by inhibiting the growth of Helicobacter pylori (two reference and 34 clinical isolates). The complex inhibited the growth of H. pylori in an MIC range of 1-8 microg mL(-1). The anti-H. pylori activity of the zinc-famotidine complex against antibiotic-resistant strains was nearly comparable to that of antibiotic-susceptible strains. The complex was found to be far less toxic than the parent drug, as demonstrated by its higher LD(50) value. In the human urease enzyme inhibition assay the complex exhibited significant inhibition. The new complex appears to be more useful in eradicating both the antibiotic-susceptible and antibiotic-resistant strains of H. pylori.

Objectives: The aim of this study was to characterize a putative novel macrolide efflux gene located in the vicinity of sul3 in porcine Escherichia coli. Methods: Five sul3-encoding E. coli isolates of porcine origin were investigated by plasmid characterization and random amplification of polymorphic DNA (RAPD) PCR. Unknown DNA adjacent to the sul3 genes was amplified using a PCR approach, followed by sequencing of the fragments. The putative macrolide efflux gene was cloned into pK18. The cloned gene was characterized by susceptibility testing by Etest in the presence and absence of efflux inhibitors. Results: Five sul3-encoding isolates, demonstrated to be unrelated by RAPD PCR, were characterized. The immediate genetic context of sul3 in five isolates was identical to that in plasmid pVP440, and in all cases, sul3 was associated with class 1 integrons. In three isolates, an open reading frame (orf2) encoding a putative protein with 38% amino acid identity to Mef(A) was found, while the two remaining isolates contained a fragment of orf2 truncated by IS26 insertion. In three of the isolates, this DNA region was demonstrated to be located on non-conjugative plasmids. When the complete orf2 was cloned, it conferred high-level resistance to erythromycin and azithromycin, and the resistance property could be partially inhibited using the efflux inhibitor Phe-Arg b-naphthylamide dihydrochloride. The gene was named mef(B). Conclusions: A new macrolide efflux protein, Mef(B), with 38% amino acid identity to Mef(A), has been characterized and represents the second member of the mef family of genes.

The incidence of antimicrobial resistance and expressed and unexpressed resistance genes among commensal Escherichia coli isolated from healthy farm animals at slaughter in Great Britain was investigated. The prevalence of antimicrobial resistance among the isolates varied according to the animal species; of 836 isolates from cattle tested only 5.7% were resistant to one or more antimicrobials, while only 3.0% of 836 isolates from sheep were resistant to one or more agents. However, 92.1% of 2480 isolates from pigs were resistant to at least one antimicrobial. Among isolates from pigs, resistance to some antimicrobials such as tetracycline (78.7%), sulphonamide (66.9%) and streptomycin (37.5%) was found to be common, but relatively rare to other agents such as amikacin (0.1%), ceftazidime (0.1%) and coamoxiclav (0.2%). The isolates had a diverse range of resistance gene profiles, with tet(B), sul2 and strAB identified most frequently. Seven out of 615 isolates investigated carried unexpressed resistance genes. One trimethoprim-susceptible isolate carried a complete dfrA17 gene but lacked a promoter for it. However, in the remaining six streptomycin-susceptible isolates, one of which carried strAB while the others carried aadA, no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. The data indicate that antimicrobial resistance in E. coli of animal origin is due to a broad range of acquired genes.

Objectives To investigate the presumed acquisition of ampicillin resistance by an Escherichia coli strain residing in the gut of an infant. Methods E. coli strains were quantified in faecal samples obtained at regular intervals from an infant followed from birth to 12 months of age and their resistance profiles were determined. beta-Lactamases were identified by isoelectric focusing and genes by PCR followed by DNA sequencing. Plasmids were characterized by restriction fragment analysis and Southern-blot hybridization, and tested for conjugative transfer. Results The infant carried two E. coli strains, termed 29A and 29B, simultaneously in the microbiota during the first month of life. All isolates of 29A were resistant to ampicillin, whereas strain 29B, which was initially ampicillin susceptible, acquired resistance following treatment of the infant with ampicillin/amoxicillin because of urinary tract infection. Acquisition of resistance by strain 29B was associated with acquisition of a bla(TEM-1b)-encoding plasmid, pNK29, which was also present in strain 29A. Transfer of plasmid pNK29 could be replicated by conjugation from strain 29A to strain 29B in vitro. Strain 29A also adapted to ampicillin treatment by mutation of the bla(TEM-1b) promoter gene to yield a higher level of resistance. Conclusions This is an unequivocal demonstration of gene transfer between two strains co-residing in the human gut, as the donor, recipient and transconjugant strains were isolated. The results suggest the dynamic adaptation by commensal bacteria in response to antibiotic treatment may occur readily.

Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae . The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples ( n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens.

The possibility that unexpressed antibiotic resistance genes are carried by bacterial genomes is seldom investigated. Potential silencing of the resistance genes bla OXA-2 , aadA1 , sul1 , and tetA carried on the plasmid pVE46 in a recent porcine isolate of Escherichia coli was investigated following oral inoculation of the strain into organic piglets. A small proportion of isolates recovered from feces did not express one or more resistance genes, despite retaining the pVE46 plasmid. Different combinations of unexpressed resistance genes were observed, and 12 representative isolates were selected for further study. Surprisingly, in most cases the resistance genes and their promoters, although not expressed, were intact, with fully wild-type sequences. Apart from four isolates exhibiting intermediate-level tetracycline resistance, no mRNA for the unexpressed genes was detected. Silencing of resistance genes was reversible at low frequencies between 10 −6 and 10 −10 . Introduction of the plasmid from silenced isolates to another strain restored expression, indicating that gene silencing was a property of the host chromosome rather than the plasmid itself. When the same recent porcine E. coli strain carrying the unrelated plasmid RP1 was inoculated into piglets, three isolates (of 9,492) that no longer expressed RP1-encoded resistance genes were recovered. As with pVE46, in most cases the coding sequences and promoter regions of these genes were found to be intact, but they were not transcribed. Such gene silencing indicates a previously unrecognized form of transcriptional control that overrides standard expression signals to shut down gene expression. These findings suggest that unexpressed resistance genes may occur in the wild and hence may have clinical implications.

Objectives Little is known of the fitness cost that antibiotic resistance exerts on wild-type bacteria, especially in their natural environments. We therefore examined the fitness costs that several antibiotic resistance elements imposed on a wild-type Escherichia coli isolate, both in the laboratory and in a pig gut colonization model. Methods Plasmid R46, Tn1 and Tn7 and a K42R RpsL substitution were separately introduced into E. coli 345-2 RifC, a rifampicin-resistant derivative of a recent porcine isolate. The insertion site of Tn1 was determined by DNA sequencing. The fitness cost of each resistance element was assessed in vitro by pairwise growth competition and in vivo by regularly monitoring the recovery of strains from faeces for 21 days following oral inoculation of organic piglets. Each derivative of 345-2 RifC carrying a resistance element was grown in antibiotic-free broth for 200 generations and the experiments to assess fitness were repeated. Results RpsL K42R was found to impose a small fitness cost on E. coli 345-2 RifC in vitro but did not compromise survival in vivo. R46 imposed a cost both before and after laboratory passage in vitro, but only the pre-passage strain was at a disadvantage in vivo. The post-passage isolate had an advantage in pigs. Acquisition of Tn7 had no impact on the fitness of E. coli 345-2 RifC. Two derivatives containing Tn1 were isolated and, in both cases, the transposon inserted into the same cryptic chromosomal sequence. Acquisition of Tn1 improved fitness of E. coli 345-2 RifC in vitro and in vivo in the case of the first derivative, but in the case of a second, independent derivative, Tn1 had a neutral effect on fitness. Conclusions The fitness impact imposed on E. coli 345-2 RifC by carriage of antibiotic resistance elements was generally low or non-existent, suggesting that once established, resistance may be difficult to eliminate through reduction in prescribing alone.

Objectives: The genetic basis of rifampicin resistance and the associated fitness cost in Enterococcus faecium were investigated. Methods: Twelve spontaneous rifampicin-resistant E. faecium mutants were selected from four parent strains recently isolated from porcine faecal material. The DNA sequence of the complete rpoB gene from the parent strains and of nucleotides -189 to +1785 from the mutants was determined from PCR amplicons. The fitness of the mutants was assessed by determining growth rate, by direct growth competition and by the ability of some of the mutants to survive in the pig intestine. Results: The rpoB genes of the parent strains diverged from each other by 1-10% and each encoded pro- teins that were 1208 amino acids in length. All mutants had a single amino acid substitution in the region implicated in rifampicin resistance in other organisms. Six mutants carried the substitution H489Y/Q, two mutants carried the substitution R492H, one mutant carried the substitution Q480H, two mutants carried the substitutions S494L and V224I, and one mutant carried the substitutions G485D and V224I. Per generation fitness costs of the mutants ranged from a gain of 2.5% to a cost of 10%. Mutants with the substitution H489Y/Q were the most fit, whereas the double mutants were the least fit. The mutant with the substitution H489Q was able to survive in the pig gut for 12 days. There was some correlation between the rifampicin MIC and fitness cost, with higher MICs being associated with higher fitness costs. Conclusions: Substitutions in RpoB are associated with rifampicin resistance in E. faecium. The fitness cost of resistance is variable and can sometimes be absent.

Background In sub-Saharan Africa there is scanty data on the causes of neonatal sepsis and antimicrobial resistance among common invasive pathogens, which might guide policy and practice. Methods This was a cross-sectional observational prevalence and aetiology study of neonates with suspected sepsis admitted to the neonatal intensive care unit, University Teaching Hospital, Lusaka, Zambia, between October 2013 and May 2014. Data from blood cultures and phenotypic antibiotic susceptibility testing were compared with multivariate analysis of risk factors for neonatal sepsis. Results Of 313 neonates with suspected sepsis, 54% (170/313) were male; 20% (62/313) were born to HIV-positive mothers; 33% (103/313) had positive blood cultures, of which 85% (88/103) were early onset sepsis (EOS). Klebsiella species was the most prevalent isolate, accounting for 75% (77/103) of cases, followed by coagulase-negative staphylococci (6% (7/103)), Staphylococcus aureus (6% (6/103)), Escherichia coli (5% (5/103) and Candida species (5% (5/103). For Klebsiella species, antibiotic resistance ranged from 96–99% for WHO-recommended first-line therapy (gentamicin and ampicillin/penicillin) to 94–97% for third generation cephalosporins. The prevalence of culture-confirmed sepsis increased from 0–39% from December 2013 to March 2014, during which time mortality increased 29–47%. 93% (14/15) neonates with late onset sepsis and 82% (37/45) with early-onset sepsis aged 4–7 days were admitted >2 days prior to onset of symptoms. Culture results for only 25% (26/103) of cases were available before discharge or death. Maternal HIV infection was associated with a reduced risk of neonatal sepsis (OR 0.46 [0.23–0.93], p=0.029). Conclusions Outbreaks of nosocomial multi-antibiotic-resistant infections are an important cause of neonatal sepsis and associated mortality. Reduced risk of neonatal sepsis associated with maternal HIV infection is counterintuitive and requires further investigation.

Hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) continue to present very significant diagnostic and management challenges. The development, introduction and use of a wider range of immunosuppressive therapies are leading to a broader spectrum of microorganisms causing HAP and VAP. The persistent clinical dilemma regarding their cause is that detection of a microorganism from a respiratory tract sample does not necessarily signify it is the causative agent of the pneumonia. The ever-increasing antibiotic resistance problem means that HAP and VAP are becoming progressively more difficult to treat. In this article, we review the cause, antimicrobial resistance, diagnosis and treatment of HAP and VAP and encapsulate recent developments and concepts in this rapidly moving field.Although the microbial causes of HAP and VAP remain at present similar to those identified in previous studies, there are marked geographical differences. Resistance rates among Gram-negative bacteria are continually increasing, and for any species, multiresistance is the norm rather than the exception. The development and introduction of rapid point-of-care diagnostics may improve understanding of the cause of HAP and VAP and has immense potential to influence the treatment and clinical outcomes in HAP/VAP, with patients likely to receive much faster, microorganism-specific treatment with obvious downstream improvements to clinical outcome and antimicrobial stewardship.We describe recent trends in aetiology of HAP and VAP and recent trends in antimicrobial resistance, including resistance mechanisms causing particular concern. The potential for novel molecular diagnostics to revolutionize the diagnosis and treatment of HAP/VAP is discussed.

The occurrence of antibiotic-resistant bacteria is an increasingly serious problem world-wide. In addition, to phenotypically resistant bacteria, a threat may also be posed by isolates with silent, but intact, antibiotic resistance genes. Such isolates, which have recently been described, possess wild-type genes that are not expressed, but may convert to resistance by activating expression of the silent genes. They may therefore compromise the efficacy of antimicrobial treatment, particularly if their presence has not been diagnosed. This chapter describes the detection of silent resistance genes by PCR and DNA sequencing. A method to detect five potentially silent acquired resistance genes; aadA, bla (OXA-2), strAB, sul1, and tet(A) is described. First, the susceptibility of the isolates to the relevant antibiotics is determined by an appropriate susceptibility testing method, such as E-test. Then the presence of the genes is investigated by PCR followed by agarose gel electrophoresis of the amplification products. If a resistance gene is detected in a susceptible isolate, the entire open-reading frame and promoter sequence of the gene is amplified by PCR and their DNA sequences obtained. The DNA sequences are then compared to those of known resistant isolates, to detect mutations that may account for susceptibility. If no mutations are detected the expression of the gene is investigated by RT-PCR following RNA extraction. The methods described here can be applied to all acquired resistance genes for which sequence and normal expression data are available.

The occurrence of antibiotic-resistant bacteria is an increasingly serious problem world-wide. In addition, to phenotypically resistant bacteria, a threat may also be posed by isolates with silent, but intact, antibiotic resistance genes. Such isolates, which have recently been described, possess wild-type genes that are not expressed, but may convert to resistance by activating expression of the silent genes. They may therefore compromise the efficacy of antimicrobial treatment, particularly if their presence has not been diagnosed. This chapter describes the detection of silent resistance genes by PCR and DNA sequencing. A method to detect five potentially silent acquired resistance genes; aadA, bla (OXA-2), strAB, sul1, and tet(A) is described. First, the susceptibility of the isolates to the relevant antibiotics is determined by an appropriate susceptibility testing method, such as E-test. Then the presence of the genes is investigated by PCR followed by agarose gel electrophoresis of the amplification products. If a resistance gene is detected in a susceptible isolate, the entire open-reading frame and promoter sequence of the gene is amplified by PCR and their DNA sequences obtained. The DNA sequences are then compared to those of known resistant isolates, to detect mutations that may account for susceptibility. If no mutations are detected the expression of the gene is investigated by RT-PCR following RNA extraction. The methods described here can be applied to all acquired resistance genes for which sequence and normal expression data are available.

The aim of this study was to evaluate the ability of an Escherichia coli with the multiple antibiotic resistance (MAR) phenotype to withstand the stresses of slaughter compared to an isogenic progenitor strain. A wild type E. coli isolate (345-2RifC) of porcine origin was used to derive 3 isogenic MAR mutants. Escherichia coli 345-2RifC and its MAR derivatives were inoculated into separate groups of pigs. Once colonisation was established, the pigs were slaughtered and persistence of the E. coli strains in the abattoir environment and on the pig carcasses was monitored and compared. No significant difference (P>0.05) was detected between the shedding of the different E. coli strains from the live pigs. Both the parent strain and its MAR derivatives persisted in the abattoir environment, however the parent strain was recovered from 6 of the 13 locations sampled while the MAR derivatives were recovered from 11 of 13 and the number of MAR E. coli recovered was 10-fold higher than the parent strain at half of the locations. The parent strain was not recovered from any of the 6 chilled carcasses whereas the MAR derivatives were recovered from 3 out of 5 (P

Summary The prevalence of antibiotic-resistant bacteria in wild animal and bird populations is largely unknown, with little consistency among the few published reports. We therefore examined intestinal bacteria from magpies (Pica pica) and rabbits (Oryctolagus cuniculus) collected in rural west Wales. Escherichia coli isolates resistant to multiple antibiotics were grown from eight of 20 magpies trapped in spring, 1999 and one of 17 in spring, 2000; the most prevalent resistance trait among these isolates was to tetracycline, but resistances to ampicillin, chloramphenicol, kanamycin, sulphonamide, tetracycline and trimethoprim were also found. Tetracycline-resistant Enterococcus spp. were found in one of 20 magpies in 1999 and three of 17 in 2000. Only one resistant E. coli isolate was detected among gut bacteria from 13 rabbits, and this strain was resistant only to tetracycline. Differences in the prevalence of resistance between bacteria from rabbits and magpies may reflect differences in diet: rabbits graze field edges, whereas magpies are omnivorous and opportunistic. The resistance genes found in E. coli isolates from magpies mostly corresponded to those common among human isolates, but those conferring tetracycline resistance were unique.

Summary Background There is a clear association between heavy antimicrobial consumption within a population and the frequent recovery of resistant bacteria, but whether a reduction in antimicrobial use can reverse this process is less clear. We investigated the effect of a national restriction of sulphonamide prescribing in the UK on the prevalence of sulphonamide resistance in Escherichia coli. Methods Consecutive clinical isolates of E coli were collected at the Royal London Hospital in 1991 and 1999. These collections, each of more than 350 isolates, were compared. Minimum inhibitory concentrations of sulphamethoxazole and eight other antimicrobials were determined. The presence and locations of sulphonamide-resistance genes were examined by PCR, plasmid extraction, Southern hybridisation, and transconjugation. Findings Despite a huge decrease in sulphonamide prescriptions (from 320 000 prescriptions per year in 1991 to 7 000 in 1999), the frequency of resistance remained high in 1999 (165/359 [46·0%] vs 143/360 [39·7%] in 1991; difference 6·2% [95% CI -0·9 to 13·3]). Integronborne sulI was present in 16·4% of isolates in 1991 and 17·5% in 1999. The prevalence of sulII increased from 26·7% in 1991 to 36·5% in 1999 (difference 9·8% [3·1 to 16·5] p=0·0046). SulII was located on large plasmids, at least some of which were conjugative multiresistance determinants. Interpretation These results show that a huge decrease in antibiotic prescribing does not necessarily reduce resistance within a useful time. The main reason seems to be the genetic linkage of the index resistance to other resistance determinants.

A key component for tackling the ever more serious antimicrobial resistance problem in Gram-negative bacteria is the introduction of rapid nucleic acid diagnostics. Successful incorporation of new diagnostic technologies has the potential benefit of improving not only patient treatment but also infection control and antimicrobial stewardship. However, there are still many hurdles to overcome, such as the complexity of resistance mechanisms in Gram-negative bacteria, the discrepancy between phenotype and genotype and the difficulty in distinguishing pathogens from background commensals. A small number of manufacturers have introduced tests to the market that concentrate partly or specifically on resistance determinants in Gram-negative bacteria. These are currently predominantly based on different types of PCR technology. The development of new technologies, such as whole-genome sequencing and the combination of MALDI-TOF with PCR, holds much promise for the introduction of improved diagnostics for the future.

Background Understanding the survival of resistance plasmids in the absence of selective pressure for the antibiotic resistance genes they carry is important for assessing the value of interventions to combat resistant bacteria. Here, several poorly explored questions regarding the fitness impact of IncP1 and IncN broad host range plasmids on their bacterial hosts are examined; namely, whether related plasmids have similar fitness impacts, whether this varies according to host genetic background, and what effect antimicrobial resistance gene silencing has on fitness. Results For the IncP1 group pairwise in vitro growth competition demonstrated that the fitness cost of plasmid RP1 depends on the host strain. For the IncN group, plasmids R46 and N3 whose sequence is presented for the first time conferred remarkably different fitness costs despite sharing closely related backbone structures, implicating the accessory genes in fitness. Silencing of antimicrobial resistance genes was found to be beneficial for host fitness with RP1 but not for IncN plasmid pVE46. Conclusions These findings suggest that the fitness impact of a given plasmid on its host cannot be inferred from results obtained with other host-plasmid combinations, even if these are closely related.