Hospital Reservoirs of Multidrug Resistant Acinetobacter Species-The Elephant in the Room!

Environmental contamination is estimated to contribute to up to 20% of all hospital acquired infections. Acinetobacter baumannii is an example of one the most prevalent opportunistic pathogens causing severe and persistent infections in immunocompromised patients. It has proven ability to form biofilms, has significant associated multi-drug resistance and is able to transfer mobile genetic elements to other clinically relevant pathogens. All of these factors point to a definite utility of A. baumannii as an indicator organism for effectiveness of decontamination regimens as well as environmental screening. There is an increased cost, both financial and clinical, associated with multi drug resistant organisms, carbapenem resistant A. baumannii. With a dearth of new antimicrobials in development, now is the time to radically transform and lead the introduction of scientifically based environmental screening and microbiological verified decontamination to control the dissemination of further resistance.

Infectious Mononucleosis: diagnosis and clinical interpretation.

EBV is the sole causative agent of the acute illness in humans described either as infectious mononucleosis (IM), or glandular fever. IM, when not clinically silent, can present in patients with at least two of the classic triad of symptoms of fever, pharyngitis, and lymphadenopathy. Challenges for the clinician arise when atypical cases present. Early, accurate and informed laboratory test results are vital for diagnosis, appropriate treatment, and management. A key challenge for the practitioner, particularly in cases where the illness can present atypically, is distinguishing bacterial tonsillitis infections from early acute IM. The ability to draw on timely, clear, and insightful laboratory results to distinguish viral from bacterial infection is vital. Correct and prompt diagnosis of IM can help prevent the unnecessary administration of antibiotics and mitigate the need for other expensive exploratory tests in cases of IM that present with splenomegaly, lymphadenopathy, or suspect haematological conditions. Good communication between the requesting clinician and those carrying out the investigative process, and between the different laboratory departments involved, is good practice and would ultimately benefit the patient. This communication will comprehensively review the aetiology, clinical presentation, and laboratory findings in IM with a view to promoting further research and so derive a standard diagnostic algorithm of the condition.

An in vitro investigation of the survival and/or growth of Campylobacter jejuni in broiler digestate from different feed types.

Campylobacter spp. is the leading cause of bacterial gastroenteritis worldwide and poultry are the primary reservoir. The aim of this study was to investigate the survival and/or growth of Campylobacter jejuni NCTC 11168 in broiler digestate prepared from commercial starter, grower and finisher feed formulations. Bolton broth and digestates were prepared, inoculated with C. jejuni NCTC 11168 (approximately 3 log10 CFU per ml) and incubated under microaerobic conditions at 42°C for 24 h. Samples were taken at t = 0 (immediately after inoculation) and every 3 h thereafter, serially diluted and plated onto mCCDA. Campylobacter jejuni grew as expected in Bolton broth (control) reaching the early stationary phase after approximately 15 h. In contrast, although bacterial concentrations were maintained for at least 9 h, none of the feed digestates supported the growth of C. jejuni, which were not detected after 15 h. It is suggested that the nutrients available in the feed digestates are not enough to support C. jejuni growth and that additional factors may be at play in the avian gastrointestinal tract.

Antimicrobial resistance of Campylobacter isolates recovered from broilers in the Republic of Ireland in 2017 and 2018: an update.

1. Campylobacteriosis is the leading cause of human bacterial gastroenteritis. Broilers are considered the most important source of human Campylobacter infection. In the 2008 European baseline survey Ireland had a 98% prevalence of campylobacter-contaminated broiler carcases. 2. Randomly-selected Campylobacter isolates (296 C. jejuni, 54 C. coli) recovered in 2017 and 2018, from Irish broiler neck skin and caeca were tested for their resistance to tetracycline, erythromycin, gentamicin, ciprofloxacin, nalidixic acid and streptomycin. 3. Overall, 45% of the Campylobacter spp. isolates tested were resistant to at least one antimicrobial. Tetracycline resistance (38%) was most prevalent in C. jejuni, followed by ciprofloxacin and nalidixic acid resistance (29%). In C. coli, resistance to ciprofloxacin and nalidixic acid (26%) was most prevalent followed by resistance to tetracycline (13%). Gentamicin resistance was undetected and resistance to streptomycin was low for C. jejuni (1%) and C. coli (4%). All C. jejuni isolates examined were erythromycin-sensitive, while 9% of C. coli isolates were erythromycin-resistant. Three multidrug-resistant C. coli isolates were recovered. 4. While antibiotic resistance rates were somewhat similar to figures reported nationally over the past 20 years, the prevalence of tetracycline resistance in C. jejuni has increased. The persistence of substantial ciprofloxacin resistance in the Irish broiler population was noteworthy, despite fluoroquinolones having been banned for growth promotion in Europe since 2006.

Carbapenemase screening in an Irish tertiary referral hospital: Best practice, or can we do better?

BACKGROUND: Carbapenems are a family of end line antibiotics with increasing levels of resistance that are a cause for concern. AIM: To ascertain whether the CPE screening programme employed in an acute tertiary hospital is fit for purpose. METHOD: We outlined the current working algorithm employed using a universal screening programme over a 26-month screening period. Rectal swabs are cultured on arrival. Those with suspicious growth are further investigated using NG-Carba 5 lateral flow tests and Vitek 2.0 sensitivity cards. These practices were compared with NHS guidelines. FINDINGS & CONCLUSIONS: In all, 53 true positives were detected from 45 patients since the screening was implemented in early 2018 (46 OXA-48, 6 KPC, 1 NDM). As the rate of screening increased, the number of positive screens decreased over time. There were a lot of similarities between the HSE guidelines and the published NHS CPE toolkit. It was evident that there is no standard practice being employed across all hospitals. Comparing the MUH to national guidelines it appears to be quicker and more effective with universal screening in place at reducing the potential contacts and identifying carriers. Cost analysis indicates that the need to confirm all positive strains in a reference lab is costly, unnecessary and time consuming. There are adequate confirmatory tests available in-house for routine positive screens. It was concluded that infection prevention and control are key to identifying and controlling possible outbreaks in a hospital setting.

Assessment of the comparability of CLSI, EUCAST and Stokes antimicrobial susceptibility profiles for Escherichia coli uropathogenic isolates.

BACKGROUND: As many clinical laboratories convert between Stokes, Clinical and Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) methods, the problem of comparing differently derived sets of antimicrobial susceptibility testing (AST) data with each other arises, owing to a scarcity of knowledge of inter-method comparability. The purpose of the current study was to determine the comparability of CLSI, EUCAST and Stokes AST methods for determining susceptibility of uropathogenic Escherichia coli to ampicillin, amoxicillin-clavulanate, trimethoprim, cephradine/cephalexin, ciprofloxacin and nitrofurantoin. METHODS: A total of 100 E. coli isolates were obtained from boric acid urine samples from patients attending GP surgeries. For EUCAST and CLSI, the Kirby-Bauer disc diffusion method was used and results interpreted using the respective breakpoint guidelines. For the Stokes method, direct susceptibility testing was performed on the urine samples. RESULTS: The lowest levels of agreement were for amoxicillin-clavulanate (60%) and ciprofloxacin (89%) between the three AST methods, when using 2017 interpretive guidelines for CLSI and EUCAST. A comparison of EUCAST and CLSI without Stokes showed 82% agreement for amoxicillin-clavulanate and 94% agreement for ciprofloxacin. Discrepancies were compounded by varying breakpoint susceptibility guidelines issued during the period 2011-2017, and through the inclusion of a definition of intermediate susceptibility in some cases. CONCLUSIONS: Our data indicate that the discrepancies generated through using different AST methods and different interpretive guidelines may result in confusion and inaccuracy when prescribing treatment for urinary tract infection.

How to: identify non-tuberculous Mycobacterium species using MALDI-TOF mass spectrometry.

BACKGROUND: The implementation of MALDI-TOF MS for microorganism identification has changed the routine of the microbiology laboratories as we knew it. Most microorganisms can now be reliably identified within minutes using this inexpensive, user-friendly methodology. However, its application in the identification of mycobacteria isolates has been hampered by the structure of their cell wall. Improvements in the sample processing method and in the available database have proved key factors for the rapid and reliable identification of non-tuberculous mycobacteria isolates using MALDI-TOF MS. AIMS: The main objective is to provide information about the proceedings for the identification of non-tuberculous isolates using MALDI-TOF MS and to review different sample processing methods, available databases, and the interpretation of the results. SOURCES: Results from relevant studies on the use of the available MALDI-TOF MS instruments, the implementation of innovative sample processing methods, or the implementation of improved databases are discussed. CONTENT: Insight about the methodology required for reliable identification of non-tuberculous mycobacteria and its implementation in the microbiology laboratory routine is provided. IMPLICATIONS: Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable, and inexpensive manner.

Three-centre evaluation of laboratory Clostridium difficile detection algorithms and the EntericBio® realtime C. difficile assay.

The comparatively high cost of laboratory detection methods for Clostridium difficile infection (CDI) coupled to a low prevalence rate has resulted in testing algorithms that use cheaper and relatively sensitive screening methods, followed by more specific confirmatory methods. The aim of this prospectively-conducted study from two centres in the UK, and one in the Republic of Ireland was to determine the efficacy of the EntericBio® realtime C. difficile Assay (EBCD) for the detection of toxigenic C. difficile in stool samples. The EBCD was compared to the in-use testing methods for Clostridium difficile (CD) detection in each centre. In the two UK centres, the EBCD was compared to the C.diff Quik Chek Complete® kit (Techlab), and discrepancies were tested further using The Xpert®C. difficile PCR assay (Cepheid) and PCR ribotyping after cultivation using the spore culture method, respectively. In the Irish centre, EBCD comparison was to an algorithm of C. DIFF CHEK™-60 test (Techlab) for screening followed by C. difficile Premier ™ Toxins A&B assay (Meridian Bioscience®) in the case of positive results; discrepancies were tested using the Xpert®C. difficile PCR assay. In a retrospective analysis of data, a total of 947 stool samples were tested, of which eight (0.8%) proved inhibitory to the EBCD assay. Of the 939 valid tests conducted, reported sensitivities of the EBCD were 94.7%, 100% and 97.9%, respectively; specificities were 99.6%, 100% and 100%, respectively; positive predictive values were 94.7%, 100% and 100%, respectively, and negative predictive values were 99.6%, 100% and 99.8%, respectively. The CD positivity rates in the current study ranged between 6.6% and 8.2%.

Towards understanding clinical campylobacter infection and its transmission: time for a different approach?

Campylobacter spp. are among the most commonly diagnosed causes of human infection. Methods for detection of the 29 campylobacter species have mainly focused on cultivation of the thermophilic species. More than 99% of clinical campylobacter isolates notified in the UK in the recent past have been from faecal samples and associated with gastroenteritis. Campylobacter enteritis notifications in temperate zones show a seasonal increase during the summer months with a sharp decrease in the winter months, a pattern which remains incompletely understood. The striking seasonality in the expression of many human genes, some concerned with inflammation and immunity, suggests a need for further study of the host regarding the temporal distribution of many human infections, including campylobacteriosis. A tendency for campylobacter to enter a non-cultivable state under adverse conditions effects a reduction in the number of isolations. A Polymerase Chain Reaction (PCR)-based screening approach for the presence of the Campylobacter genus and followed by speciation has provided some insight into the limitations of cultivation for campylobacter, also allowing the discovery of new species. The increased sensitivity of the PCR-based approach over culture-based methods may make it difficult for the laboratory to differentiate asymptomatic campylobacter carriage from clinical campylobacter infection in non-sterile body sites. Campylobacter infection depends on a combination of host factors, and on acquisition of a suitably virulent strain with a tropism for human epithelium. The possibility of persistence of campylobacter in a viable but non-culturable latent form in the human body may also require further investigation. The scope of this review includes a discussion of current methods for diagnosing acute campylobacter infection and for detecting campylobacter in water and foodstuffs. The review also questions the prevailing view that poultry is the most common source of campylobacteriosis.

Vancomycin-resistant enterococci carriage in an acute Irish hospital.

BACKGROUND: Ireland has been shown to have the highest rate of vancomycin-resistant enterococci (VRE) in cases of bacteraemia in Europe, according to a report in 2014 from the European Antimicrobial Resistance Surveillance System Network. AIM: To investigate the prevalence of VRE gut colonization in a cohort of patients in 2014 at Cork University Hospital (CUH) by performing a cross-sectional study using faecal samples submitted to the microbiology laboratory for routine investigation from both hospital inpatients and community-based patients. METHODS: Faeces were examined for VRE colonization using selective cultivation, antimicrobial susceptibility testing, and speciation using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. All VRE isolates were evaluated by molecular means for resistance determinants, type, and Insertion Sequence 16 as an indicator of Clonal Complex 17 (CC17). FINDINGS: From the 350 specimens investigated, 67 (19.1%) specimens were positive for VRE [95% confidence interval (CI): 15.0-23.2]. The prevalence of VRE colonization among CUH patients tested in this study (N = 194) was 31.4% (95% CI: 24.7-38.1). By contrast, the general practitioner patient samples (N=29) showed a prevalence of 0%, whereas 22.2% of samples from other hospitals (N=27) were positive for VRE. All isolates were Enterococcus faecium (VREfm) and were indicated to contain CC17, though with considerable heterogeneity among the isolates. CONCLUSION: This high prevalence goes some way towards providing an explanation for the current high rates of VRE bacteraemia in Ireland, as well as highlighting the benefits of screening and enhanced infection control practices by all hospitals to control the high rates of VRE observed.

Mycobacterium diagnostics: from the primitive to the promising.

The field of clinical microbiology has been revolutionised by genomic and proteomic methods, which have facilitated more rapid diagnosis and characterisation of infection in many cases. In contrast, mycobacteriological evolution has tended to retain the traditional methods of smear microscopy for detection of acid-fast bacilli to indicate mycobacteria, along with culture, and in synergy with more modern molecular methods. Thus, efforts have been focused on reducing the time to diagnosis of infection, while increasing the amount of diagnostic information available, including more definitive speciation, and more rapid susceptibility test results. Although smear microscopy remains a mainstay for the laboratory-based diagnosis of mycobacterial infection, molecular testing has vastly reduced the time needed for identification of Mycobacterium tuberculosis in particular, when compared with traditional culture-based techniques. Molecular methods may also yield antimicrobial susceptibility results through testing for the most common resistance-inducing mutations to some of the antimicrobial agents of choice. However, the diversity of resistance mutations already characterised suggests that these currently-available molecular detection systems should be accompanied by culture-based susceptibility testing. This review compares the efficacy of microscopic, phenotypic, proteomic and genotypic methods available for mycobacterial diagnosis. The diversity of methods currently in use reflects the complexity of this area of diagnostic microbiology.

A post-antibiotic era looms: can plant natural product research fill the void?

Antimicrobial resistance is increasing among certain pathogenic bacteria to the extent that treatment efficacy is no longer always assured. According to the CDC, as few as six new antibiotics have been released for use over the past 30 years. Resistance has already been observed to each of these. Eleven plant natural products have been approved for therapeutic use during the same period--none of them being antimicrobial agents. We have learned through experience that some microorganisms will inevitably overcome antibiotic treatment in certain situations, and then spread. It is clear that the rate of new antimicrobial development is insufficient to meet our current and future needs, which should be addressed in order to guarantee the effective future of antimicrobial chemotherapy. However, in recent years there has been an increase in the number of peer-reviewed reports of antimicrobial efficacy among plant-derived secondary metabolites. A limitation with these reports is the wide range of modified in vitro methods used to determine antimicrobial efficacy of these products, showing an absence of the type of standardisation that is the norm when testing the efficacy of single- or combined-agent conventional antimicrobials in the laboratory, thereby making inter-study comparison difficult. Overall, despite the large diversity in preparation and testing strategies used currently for natural product plant-derived antimicrobials, our investigations suggest that the field shows promise in the provision of novel antimicrobial agents, even as exemplified by our selected example, Inula helenium (Elecampane).

Development of nalidixic acid amphotericin B vancomycin (NAV) medium for the isolation of Campylobacter ureolyticus from the stools of patients presenting with acute gastroenteritis.

Recently, Campylobacter ureolyticus has been detected for the first time in the faeces of patients with acute gastroenteritis using polymerase chain reaction (PCR) techniques. Cultural isolation of C. ureolyticusis is not possible using the established selective methods for the isolation of thermophilic Campylobacter spp. from faeces. The aim of the current study is to develop a new selective medium capable of isolating C. ureolyticus from faecal samples. The newly-developed medium consists of Anaerobe Basal Agar with 10 g/L additional agar, 2 g/L sodium formate and 3 g/L sodium fumarate dibasic, to which 10 mg/L nalidixic acid, 10 mg/L amphotericin B and 20 mg/L vancomycin (NAV) are added as selective agents. Validation studies have shown that this experimental selective medium completely inhibits growth of Candida spp. and of Enterococcus spp. and permits reduced growth of selected coliforms and Proteus spp. Growth of Campylobacter ureolyticus on NAV medium is optimal in anaerobic and enriched hydrogen atmospheres. Additionally, an overnight enrichment step using Bolton broth to which 2 g/L sodium formate, 3 g/L sodium fumarate dibasic and the NAV supplement are added, in place of the commercial Bolton broth supplement, allows improved recovery of C. ureolyticus from patients' faeces.

Molecular-based mycobacterial identification in a clinical laboratory setting: a comparison of two methods.

Many mycobacterial species are pathogenic to humans, with infection occurring worldwide. Infection with Mycobacterium tuberculosis is a well-described global phenomenon, but other mycobacterial species are increasingly shown to be the cause of both pulmonary and extrapulmonary infection and are managed differently from M. tuberculosis infection. Rapid and accurate differentiation of mycobacterial species is, therefore, critical to guide timely and appropriate therapeutic and public health management. This study evaluates two commercially available DNA strip assays, the Genotype Common Mycobacteria (CM) assay (Hain Lifescience, Nehren, Germany) and the Speed-oligo Mycobacteria assay (Vircell, Spain) for their usefulness in a clinical laboratory setting. Both assays were evaluated on 71 clinical mycobacterial isolates, previously identified using Gen-Probe AccuProbe and through a UK mycobacteriology reference laboratory, as well as 29 non-mycobacterial isolates. Concordant results were obtained for 98% of isolates using both assays. The sensitivity was 97% (95% confidence interval [CI]: 93.3-100%) for the CM assay and 98.6% (95% CI: 95.9-100%) for the Speed-oligo assay. Overall, both assays proved to be useful tools for rapid and sensitive mycobacterial species identification, although interpretation of results was easier with the CM assay. Finally, results were available within one day, compared to current identification times which range between seven days and four weeks.

Molecular-based detection of non-culturable and emerging campylobacteria in patients presenting with gastroenteritis.

From January 2009 to May 2010, 436 faecal samples from patients with diarrhoeal illness in Southern Ireland were identified as Campylobacter genus-positive by an automated multiplex PCR; however, 204 (46·8%) of these samples were culture-negative for campylobacters. A combination of Campylobacter-specific uniplex PCR and 16S rRNA sequencing confirmed the presence of Campylobacter DNA in 191 (93·6%) of the culture-negative samples. Species-specific PCR identified C. jejuni (50·7%) C. ureolyticus (41%) and C. coli (5·7%) as the most prevalent species while C. fetus, C. upsaliensis, C. hyointestinalis and C. lari accounted for 10% of culture-negative samples; mixed Campylobacter spp. were detected in 11% of samples. We conclude that non-culturable Campylobacter spp. are responsible for a considerable proportion of human enteritis and the true incidence of infection is likely to be significantly underestimated where conventional Campylobacter culture methods are used in isolation.

Validation of a norovirus multiplex real-time RT-PCR assay for the detection of norovirus GI and GII from faeces samples.

Norovirus is a leading cause of infectious non-bacterial gastroenteritis. The virus is highly contagious and has multiple modes of transmission, presenting a growing challenge to hospital-based healthcare. In this study, a total of 120 stool samples are tested for the presence of norovirus GI and GII by the Roche two-step Lightcycler 2.0 assay incorporating primers and probes produced by TIB Molbiol, and the results are compared with results from the National Virus Reference Laboratory. The Roche/TIB Molbiol assay produced 51 positive results and 69 negative results. Discrepancy analysis was performed for six conflicting results using a second real-time polymerase chain reaction (PCR) assay (Roche/TIB Molbiol) and this confirmed that four of the five discrepant positive results were true positives. A single discrepant negative result generated by the Roche assay remained negative using the second assay. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated to be 98%, 98.6%, 98.0% and 98.6%, respectively. Melting curve analysis was used to differentiate genogroups I and II and this showed that 92% of strains belonged to genogroup II.

Emergence of group B Streptococcus serotype IV in women of child-bearing age in Ireland.

This study determined the carriage rate and serotype distribution of group B Streptococcus (GBS) in women of child-bearing age in the southern region of Ireland. A total of 2000 vaginal swabs collected in two periods in 2004 and 2006 were examined and revealed a GBS carriage rate of 16·1%. Serotyping of isolates showed that serotypes Ia, II, III, IV, and V were the most prevalent. A high prevalence of serotype IV was found, increasing from 7·6% to 15·2% between 2004 and 2006. Random amplified polymorphic DNA analysis demonstrated considerable genetic heterogeneity in the serotype IV isolates. This serotype should be considered for inclusion in potential vaccines for use in Ireland.