- Volume 60, Issue 6, 2011

AmpC β-lactamases (BlaAmpC) are an emerging group of antimicrobial resistance determinants. The lack of an agreed BlaAmpC detection method hinders investigation of their epidemiology and understanding of their clinical significance. This study compared the sensitivity and specificity of phenotypic methods of BlaAmpC detection in a collection of 246 Enterobacteriaceae with a diverse range of β-lactam resistance profiles. The BlaAmpC screening methods evaluated were based on cephamycin, ceftazidime and cefepime susceptibility. These were compared with BlaAmpC screening using conventional ESBL detection methods. The confirmatory methods evaluated were biologically based assays, inhibitor-based assays, an AmpC Etest and a rapid chromogenic assay. A multiplex nucleic acid amplification test and the three-dimensional enzyme extraction assay were used as reference methods. BlaAmpC activity was present in 74 isolates. The majority of the enzymes were plasmid-encoded and belonged to the CMY, DHA and EBC families. The screening methods had sensitivities between 47 and 99 % and specificities of 45–95 %. The performance of confirmatory tests varied widely, ranging in sensitivity from 19 % to 97 % and in specificity from 88 % to 100 %. Only the Tris-EDTA and MAST ID D68C disc tests had a sensitivity and a specificity above 90 %. Further investigation is needed to establish the most suitable enzyme substrates, inhibitor types, inhibitor concentrations and interpretative cut-offs in order to refine the inhibitor-based methods. A simple disc-based protocol using cefoxitin non-susceptibility as a screening tool, followed by the Tris-EDTA method for confirmation, detects BlaAmpC activity with 95 % sensitivity and 98 % specificity.

Novel real-time PCR assays targeting the Bordetella pertussis insertion sequence IS481, the toxin promoter region and Bordetella parapertussis insertion sequence IS1001 were designed. PCR assays were capable of detecting ≤10 copies of target DNA per reaction, with an amplification efficiency of ≥90 %. From September 2003 to December 2009, per-nasal swabs and nasopharyngeal aspirates submitted for B. pertussis culture from patients ≤1 month to >15 years of age were examined by real-time PCR. Among 1324 patients, 76 (5.7 %) were B. pertussis culture positive and 145 (10.95 %) were B. pertussis PCR positive. Of the B. pertussis PCR-positive patients, 117 (81 %) were aged 6 months or less. A total of 1548 samples were examined, of which 87 (5.6 %) were culture positive for B. pertussis and 169 (10.92 %) were B. pertussis PCR positive. All culture-positive samples were PCR positive. Seven specimens (0.5 %) were B. parapertussis culture positive and 10 (0.8 %) were B. parapertussis PCR positive, with all culture-positive samples yielding PCR-positive results. A review of patient laboratory records showed that of the 1324 patients tested for pertussis 555 (42 %) had samples referred for respiratory syncytial virus (RSV) testing and 165 (30 %) were positive, as compared to 19.4 % of the total 5719 patients tested for RSV in this period. Analysis of the age distribution of RSV-positive patients identified that 129 (78 %) were aged 6 months or less, similar to the incidence observed for pertussis in that patient age group. In conclusion, the introduction of the real-time PCR assays for the routine detection of B. pertussis resulted in a 91 % increase in the detection of the organism as compared to microbiological culture. The incidence of infection with B. parapertussis is low while the incidence of RSV infection in infants suspected of having pertussis is high, with a similar age distribution to B. pertussis infection.

The aim of this research was to analyse the resistance patterns and characterize the distribution and genetic content of resistance integrons within Enterobacter cloacae complex strains originating from hospitalized patients. The strains were included in the E. cloacae complex study following sequence analysis of the hsp60 gene. The determination of resistance towards eight classes of antimicrobials was followed by PCR detection of integrons and analyses of the size and sequences of their variable parts. The majority of 69 clinical strains of the E. cloacae complex were identified as Enterobacter hormaechei. They were isolated from a variety of samples, including urine, wounds, blood and stools. The remaining isolates belonged to E. cloacae clusters III and IV, E. cloacae subsp. cloacae and Enterobacter kobei. Fifty-two isolates (75.4 %) were resistant to more than three unrelated antibiotics. The resistance for each antibiotic, except imipenem, was significantly associated with the presence of integrons. Class 1 integrons were detected in 55 % of isolates: 63.3 % of ‘E. hormaechei subsp. steigerwaltii’, 50 % of E. cloacae cluster III, 40 % of ‘E. hormaechei subsp. oharae’, 33 % belonging to E. cloacae cluster IV and 20 % of ‘E. hormaechei subsp. hormaechei’ were intI1-positive. All of the integrons were located on transferable genetic elements. The transferred resistance primarily included that to aminoglycosides, ticarcillin, piperacillin, sulfamethoxazole, trimethoprim and tetracycline. Sequence analysis of the variable regions of integrons identified two groups of genes: those encoding aminoglycoside adenylotransferases responsible for resistance to aminoglycosides, and dfr cassettes conferring resistance to trimethoprim. Integrons of the E. cloacae complex showed limited variability of genes encoding resistance to therapeutics and were stable in structure with the following cassette arrays: dfrA12-orfF-aadA2, aadB-aadA2, dfrA1-aadA1 and aacA4-aadA1. Hospital-dependent differences in type and arrays of gene cassettes were observed, which seemed to be conserved and not liable to changes.

Anaplasma phagocytophilum is an obligately intracellular bacterium and is the causative agent of human granulocytic anaplasmosis (HGA), an emerging and major tick-borne disease in the USA and other parts of the world. This study showed that the prenylation inhibitor manumycin A effectively blocked A. phagocytophilum infection in host cells (HL-60 or RF/6A cells). A. phagocytophilum infection activated extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase in host cells, and manumycin A treatment reduced ERK activation in A. phagocytophilum-infected host cells. As ERK activation is required for A. phagocytophilum infection, we examined whether manumycin A inhibited the bacteria directly or through host ERK signalling. Treatment of A. phagocytophilum alone with manumycin A significantly reduced the bacterial infectivity of host cells and bacterial viability in the absence of host cells, whereas pre-treatment of host cells did not inhibit bacterial infection in host cells. The inhibitory effect of manumycin A on A. phagocytophilum infection in host cells was achieved even at a concentration 100 times lower than that required for effective inhibition of mammalian cell signalling. These results suggested that manumycin A directly inactivates the bacterium, resulting in reduced infection and ERK1/2 activation. Thus, the manumycin group of drugs may have a therapeutic potential for HGA.

The VITEK 2 automated system (bioMérieux) is one of the most widely used instruments in clinical microbiology laboratories for the identification and evaluation of the susceptibility profiles of bacteria including the detection of extended-spectrum β-lactamases (ESBLs) produced by Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca. Currently, the Clinical and Laboratory Standards Institute recommends the use of ESBL confirmatory tests in addition to standard susceptibility testing. In order to evaluate the accuracy of VITEK 2-positive results regarding clinical isolates of E. coli (n = 110) and K. pneumoniae (n = 72), four additional ESBL detection systems were compared: the Phoenix Automated Microbiology System (BD Diagnostic Systems) and the MicroScan WalkAway-96 System (Dade Behring), and two manual systems as confirmatory tests, the Etest (AB Biodisk) and double disc diffusion (DDS) test. Epidemiological data regarding the tested strains were also collected and their susceptibility phenotypes were determined. The four methods resulted in concordant results for 126 of the 182 strains. However, the different tests displayed distinct results: the VITEK 2 system was in disagreement in 23.9 % of cases with DDS, in 15.3 % with Etest, in 23 % with the MicroScan WalkAway-96 System and in 23.6 % with the Phoenix Automated Microbiology System. Epidemiological data indicated that the majority of ESBL-positive E. coli strains were isolated from patients admitted to internal medicine wards (72.7 %), whilst K. pneumoniae ESBL-positive isolates were equally distributed between internal medicine wards (45.8 %) and intensive care units (45.8 %). Most of these strains were isolated from urine. In contrast to ESBL-negative isolates, the ESBL-positive strains displayed multiple drug resistance, namely to quinolones, aminoglycosides and trimethoprim–sulfamethoxazole. No significant resistance to carbapenems was detected. Overall, this study demonstrates the need for a confirmatory test following positive ESBL detection with the VITEK 2 system (panel AST-037), which appears to yield a large number of false-positive results.

Between January 2007 and April 2009, rotavirus (RV)-positive stool samples from 238 children with acute gastroenteritis, seen at Children’s Hospital of Michigan in Detroit, USA, were collected and RV genotyping was performed. G and P genotypes were determined by RT-PCR and nucleotide sequencing was conducted on selected G9 and P[6] strains. Correlation between the severity of gastroenteritis episode and the infecting G genotype was done using a 14-point scoring system. The predominant G genotype was G9 (39.5 %), followed by G1 (35.3 %) and G4 (15.5 %), while P[8] was the most prevalent P genotype (66.5 %), followed by P[4] (21.9 %) and P[6] (11.2 %). The gene combinations G1P[8] and G9P[8] were the most prevalent (21.4 % and 20.6 %, respectively), followed by G4P[8] (13 %) and G9P[6] (8.8 %). Immunization data showed that only 17/238 (7.1 %) children received ≥one dose of RV vaccine (the pentavalent vaccine RotaTeq or the monovalent vaccine Rotarix) and that 10/17 were infected with G4P[8] strains. Severity of RV gastroenteritis episodes was not related to the infecting G genotype. Our results suggest a high proportion of genotype G9 strains in combination with P[8], P[6] and P[4] specificity circulating in the metropolitan Detroit area. While the protective efficacy of the RV vaccines has been demonstrated against G9P[8] strains, the level of cross-protection offered by the vaccines against G9 strains with P[6] and P[4] genotypes in the Detroit paediatric population remains to be determined.

Cytomegalovirus (CMV) enters latency following primary infection and can subsequently reactivate. Reinfection with a different viral strain can also occur. During these events, CMV is shed in bodily fluids. This study examined correlates of CMV shedding in specimens obtained from the HIV Epidemiology Research Study, a multicenter cohort study of US women with or at high risk for human immunodeficiency virus (HIV) infection. Among the women studied, 91.4 % (911/997) were CMV IgG seropositive. Of these women, 2.7 % (25/911) were CMV IgM seropositive. CMV DNA was detected via real-time PCR more frequently in cervicovaginal lavage (CVL) specimens (55/764, 7.2 %) than in peripheral blood mononuclear cells (PBMCs) (26/897, 2.9 %). CMV viral loads in 1 ml CVL (median 534; mean 2598; range = 40–74 844) were higher than in 106 PBMCs (median 264; mean 1287; range = 35–13 250). CMV DNA in PBMCs was associated with HIV seropositivity [odds ratio (OR) 13.5; 95 % confidence interval (CI) 1.8–100], increasing HIV viral load (P<0.001 for trend), decreasing CD4 cell counts (P<0.001 for trend) and CMV DNA in CVL (OR 26; 95 % CI 10.7–64). CMV DNA in CVL specimens was associated with CMV IgM seropositivity (OR 4.3; 95 % CI 1.5–12.3), HIV seropositivity (OR 7.3; 95 % CI 2.6–20), increasing HIV viral load (P<0.001 for trend) and decreasing CD4 cell counts (P<0.001 for trend). The positive predictive value of CMV IgM seropositivity for CMV DNA shedding in either PBMCs or CVL was 20 %. In summary, CMV shedding in CVL and PBMCs was highly correlated with each other and with markers of immune suppression.

Chlamydia psittaci is an obligately intracellular Gram-negative bacterium causing respiratory disease (chlamydiosis) or asymptomatic carriage in birds. C. psittaci is a zoonotic agent causing psittacosis or parrot fever in humans. Vertical and/or horizontal transmission via eggs might have serious repercussions on the C. psittaci infection status of poultry flocks and thus on zoonotic risk for all workers along the poultry supply chain. We therefore studied the presence of C. psittaci in a hatchery. In addition, we examined all (n = 4) employees of the hatchery to evaluate the zoonotic risk. We could not detect C. psittaci on either eggshells or eggshell membranes. However, C. psittaci isolates of different outer-membrane protein A (ompA) genotypes were cultured from the air of both turkey (genotypes A and C) and chicken (genotype D) hatching chambers. Zoonotic transmission occurred in all employees and a mixed infection with up to three different genotypes (A, D and C), also found in air samples, was discovered. Diagnostic monitoring and reporting of C. psittaci infections in poultry workers should be promoted. Additionally, an efficient veterinary vaccine and information campaigns on zoonotic risk and preventive measures against C. psittaci transmission would be beneficial to public health.

Leech therapy is currently considered to be of high therapeutic value in medicine. However, feeding leeches with fresh animal blood during the maintenance and reproduction phase bears the risk of transmission of zoonotic viruses to the patient. We hypothesize that this would be abolished by subjecting leeches to quarantine measures prior to use. The required duration of quarantine would depend on the maximum survival time of pathogens in contaminated leeches. In order to be able to estimate this survival time reliably, experiments were conducted with enveloped and non-enveloped mammalian viruses possessing either RNA or DNA. Leeches were fed porcine blood contaminated with bovine parvovirus (BPV), feline calicivirus (FCV), equine arteritis virus (EAV) and equine herpesvirus type 1 (EHV-1) and kept in aquaria at 10 °C. From week 6 after feeding onwards, some leeches were held at 30 °C. Before feeding and at different time points thereafter, blood samples were taken from the leeches to determine residual virus infectivity. Prototype mammalian viruses were able to survive in inoculated leeches for considerable periods of time. When leeches were kept at 10 °C throughout, reisolation of infectious virus from the leeches’ abdominal cavity blood was no longer possible at 23 (FCV), 23 (EAV), 27 (EHV-1) and 29 (BPV) weeks after inoculation. Shifting the temperature to 30 °C in week 6 slightly reduced the duration of detection of infectious viruses to 15 (EAV and EHV-1), 21 (FCV) and 27 (BPV) weeks. These data indicate that the ability of mammalian viruses to survive in leeches theoretically poses a possible risk for patients unless adequate precautionary measures are adopted. Application of a quarantine period, e.g. 31 weeks (i.e. including an additional safety period) at 10 °C, may be a suitable measure to significantly decrease this risk.

We describe associations between death from invasive pneumococcal disease (IPD) and particular serogroups and sequence types (STs) determined by multilocus sequence typing (MLST) using data from Scotland. All IPD episodes where blood or cerebrospinal fluid (CSF) culture isolates were referred to the Scottish Haemophilus, Legionella, Meningococcal and Pneumococcal Reference Laboratory (SHLMPRL) from January 1992 to February 2007 were matched to death certification records by the General Register Office for Scotland. This represented 5959 patients. The median number of IPD cases in Scotland each year was 292. Deaths, from any cause, within 30 days of pneumococcal culture from blood or CSF were considered to have IPD as a contributing factor. Eight hundred and thirty-three patients died within 30 days of culture of Streptococcus pneumoniae from blood or CSF [13.95 %; 95 % confidence interval (13.10, 14.80)]. The highest death rates were in patients over the age of 75. Serotyping data exist for all years but MLST data were only available from 2001 onward. The risk ratio of dying from infection due to particular serogroups or STs compared to dying from IPD due to all other serogroups or STs was calculated. Fisher’s exact test with Bonferroni adjustment for multiple testing was used. Age adjustment was accomplished using the Cochran–Mantel–Haenszel test and 95 % confidence intervals were reported. Serogroups 3, 11 and 16 have increased probability of causing fatal IPD in Scotland while serogroup 1 IPD has a reduced probability of causing death. None of the 20 most common STs were significantly associated with death within 30 days of pneumococcal culture, after age adjustment. We conclude that there is a stronger association between a fatal outcome and pneumococcal capsular serogroup than there is between a fatal outcome and ST.

Millions of intravaginal rings (IVRs) are used by women worldwide for contraception and for the treatment of vaginal atrophy. These devices also are suitable for local and systemic sustained release drug delivery, notably for antiviral agents in human immunodeficiency virus pre-exposure prophylaxis. Despite the widespread use of IVRs, no studies have examined whether surface-attached bacterial biofilms develop in vivo, an important consideration when determining the safety of these devices. The present study used scanning electron microscopy, fluorescence in situ hybridization and confocal laser scanning microscopy to study biofilms that formed on the surface of IVRs worn for 28 days by six female pig-tailed macaques, an excellent model organism for the human vaginal microbiome. Four of the IVRs released the nucleotide analogue reverse transcriptase inhibitor tenofovir at a controlled rate and the remaining two were unmedicated. Large areas of the ring surfaces were covered with monolayers of epithelial cells. Two bacterial biofilm phenotypes were found to develop on these monolayers and both had a broad diversity of bacterial cells closely associated with the extracellular material. Phenotype I, the more common of the two, consisted of tightly packed bacterial mats approximately 5 µm in thickness. Phenotype II was much thicker, typically 40 µm, and had an open architecture containing interwoven networks of uniform fibres. There was no significant difference in biofilm thickness and appearance between medicated and unmedicated IVRs. These preliminary results suggest that bacterial biofilms could be common on intravaginal devices worn for extended periods of time.