Antibiotic susceptibility testing and species identification of Nocardia isolates: a retrospective analysis of data from a French expert laboratory, 2010–2015

Objectives

 Nocardia, a Gram-positive bacterium, is responsible for rare and severe infections. Accurate microbiological data are essential to guide antibiotic treatment. Our primary objective was to describe species identification and results of antimicrobial susceptibility testing (AST) for Nocardia isolates analysed over a 6-year period. Secondary objectives were to study temporal trends in species distribution and AST results.

Clinical and microbiological characteristics of Nocardiosis including those caused by emerging Nocardia species in Taiwan, 1998–2008

The genus of Nocardia is rapidly expanding and the species distribution varies with different geographical locations. We retrospectively reviewed the laboratory records of the bacteriology laboratory at National Taiwan University Hospital from January 1998 to June 2008 to identify patients with nocardiosis. During the study period, 164 isolates of Nocardia spp. were identified from 134 patients but only 113 patients had Nocardia infection. Nocardia brasiliensis (n = 54) was the most common pathogen, followed by N. asteroides (n = 36), N. farcinica (n = 7), N. flavorosea (n = 4), N. otitidiscaviarum (n = 3), N. nova (n = 3), N. beijingensis (n = 2) and one each of N. puris, N. jinanensis and N. takedensis. The major types of infection were cutaneous infection (56.6%), pulmonary infection (33.6%) and disseminated infection (7.1%). Eighty-eight patients received sulfonamide-containing antibiotic and eight of 100 patients with available data on outcomes died during the episode of nocardiosis. In conclusion, the clinical and microbiological manifestations of Nocardiosis vary with the different Nocardia species. Accurate identification of the species is crucial to make the diagnosis.     

Nocardiosis in Quebec,Canada, 1988–2008

Nocardia is an uncommon pathogen, but immunosuppression, its main risk factor, is becoming more frequent. We aimed to evaluate changes in the annual incidence of nocardiosis and in the susceptibility profile of its aetiological agents. Demographic data were analysed for all isolates of Nocardia forwarded to the provincial public health laboratory of Quebec, Canada during the last two decades. Population incidence could be measured from 1997 onwards. Resistance patterns were analysed for those isolates selected for in vitro susceptibility testing. Throughout Quebec, 575 incident cases were identified between 1997 and 2008. The annual incidence of Nocardia infection/colonization increased from 0.33 (1997–1998) to 0.87 (2007–2008) per 100 000 inhabitants (p 0.001). In a small subset of patients for whom detailed clinical information was available, 59% of isolates corresponded to genuine infections. Nocardia farcinica predominated in specimens representing invasive infections (blood, brain, lung or pleural aspirates). Isolates were often non‐susceptible to several antimicrobials, with the exception of amikacin and linezolid. Overall, 43% of 157 isolates were non‐susceptible to trimethoprim–sulphamethoxazole. In conclusion, Nocardia infection/colonization remains rare. However, from 1997–1998, a progressive increase in incidence was noted in the province of Quebec. In regions such as ours, where a substantial proportion of invasive isolates are non‐susceptible in vitro to trimethoprim–sulphamethoxazole, the latter may no longer be the empirical treatment of choice in immunosuppressed and severely ill patients with nocardiosis.     

A PCR-Based Intergenic Spacer Region-Capillary Gel Electrophoresis Typing Method for Identification and Subtyping of Nocardia Species

While 16S rRNA sequence-based identification of Nocardia species has become the gold standard, it is not without its limitations. We evaluated a novel approach encompassing the amplification of the Nocardia 16S-23S rRNA intergenic spacer (IGS) region followed by fragment analysis by capillary gel electrophoresis (CGE) of the amplified product for species identification of Nocardia. One hundred forty-five Nocardia isolates (19 species) and four non-Nocardia aerobic actinomycetes were studied. Reproducibility testing was performed in a subset (21%) of isolates. Ninety-five different electropherograms were identified, with heterogeneity within species being a general observation. Among common Nocardia species (e.g., Nocardia cyriacigeorgica, N. nova, N. farcinica), 2 or 3 dominant electropherogram subgroups were typical. While only a minority (8/19; 42%) of the different Nocardia species contained isolates displaying unique fragment sizes that were predictive of a particular species, virtually all isolates (142/145; 98%) could be assigned to the correct species using IGS-CGE typing based on the number and size of amplified fragments. The median number of fragments for each isolate was 2 (range, 1 to 5) with only a minority (17%) having a single fragment detected. The majority (93%) of amplified fragments were between 408 and 461 bp. The technique was also non-operator dependent, highly reproducible, and quicker and less expensive than 16S sequencing. In summary, PCR-based IGS-CGE typing is relatively simple, accurate, reproducible, and cost-effective and offers a potential alternative to 16S rRNA sequencing for identifying and subtyping Nocardia isolates.     

Review of nocardial infections in France 1987 to 1990

On the basis of the numbers ofNocardia strains referred to the National Reference Center for Mycoses and Antifungal Agents (NRC), Institut Pasteur, Paris, in the period from 1987 to 1990, it was estimated that between 150 and 250 cases of nocardiosis are diagnosed in France each year. A total of 63 clinical isolates were referred to the NRC and identified asNocardia asteroides (66.7 %),Nocardia farcinica (23.8 %),Nocardia brasiliensis (3.2 %),Nocardia otitidiscaviarum (4.8 %) andNocardia carnea (1.5 %).Nocardia asteroides accounted for 71.4 % of pulmonary infections, 80.0 % of central nervous system infections and 80.0 % of systemic infections. Patients infected withNocardia farcinica died in 57.1 % of cases, compared with 17.6 % of patients infected withNocardia asteroides. Corticosteroid therapy represented a significant factor in mortality. Isolates ofNocardia asteroides revealed variable resistance, whereas isolates ofNocardia farcinica were resistant to most antimicrobial agents. Only amoxicillin/clavulanic acid, imipenem, cefoxitin, kanamycin, amikacin, minocycline and vancomycin showed activity against both species. Nocardiosis caused byNocardia farcinica may be a growing problem because of the relatively high incidence in AIDS patients and the resistance of this species to most antimicrobial agents.     

secA1 Gene Sequence Polymorphisms for Species Identification of Nocardia Species and Recognition of Intraspecies Genetic Diversity

Sequence analysis of the Nocardia essential secretory protein SecA1 gene (secA1) for species identification of 120 American Type Culture Collection (ATCC) and clinical isolates of Nocardia (16 species) was studied in comparison with 5′-end 606-bp 16S rRNA gene sequencing. Species determination by both methods was concordant for all 10 ATCC strains. secA1 gene sequencing provided the same species identification as 16S rRNA gene analysis for 94/110 (85.5%) clinical isolates. However, 40 (42.6%) isolates had sequences with <99.0% similarity to archived secA1 sequences for the species, including 29 Nocardia cyriacigeorgica (96.6 to 98.9% similarity) and 4 Nocardia veterana (91.5 to 98.9% similarity) strains. Discrepant species identification was obtained for 16 (14.5%) clinical isolates, including 13/23 Nocardia nova strains (identified as various Nocardia species by secA1 sequencing) and 1 isolate each of Nocardia abscessus (identified as Nocardia asiatica), Nocardia elegans (Nocardia africana), and Nocardia transvalensis (Nocardia blacklockiae); both secA1 gene sequence analysis and deduced amino acid sequence analysis determined the species to be different from those assigned by 16S rRNA gene sequencing. The secA1 locus showed high sequence diversity (66 sequence or genetic types versus 40 16S rRNA gene sequence types), which was highest for N. nova (14 secA1 sequence types), followed by Nocardia farcinica and N. veterana (n = 7 each); there was only a single sequence type among eight Nocardia paucivorans strains. The secA1 locus has potential for species identification as an adjunct to 16S rRNA gene sequencing but requires additional deduced amino acid sequence analysis. It may be a suitable marker for phylogenetic/subtyping studies.Nocardia spp. are Gram-positive saprophytic bacteria capable of causing suppurative infections, including pulmonary, cutaneous, central nervous system, and disseminated diseases. To date, approximately 90 species have been described (NCBI taxonomy for Nocardia [http://www.ncbi.nlm.nih.gov/Taxonomy/]; http://www.bacterio.cict.fr/n/nocardia.html), at least 33 of which have been implicated in human disease (2). Identification of clinical isolates to the species level is important to characterize associated disease manifestations and to predict antimicrobial susceptibility and for epidemiological and ecological purposes (2, 17).Because of the difficulty of identifying Nocardia isolates by standard phenotypically based methods and the inability of such methods to identify novel species (2, 17), various nucleic acid amplification methods targeting conserved Nocardia gene regions have been proposed to provide accurate species determination. Of these, sequence analysis of the 16S rRNA gene has become the gold standard for definitive species identification (2, 5, 6, 8, 19). Certain closely related species, however, may not be distinguished by this method due to insufficient interspecies polymorphisms within the 16S rRNA gene sequences (2, 5, 14). Other practical limitations include potential misidentifications as a result of multiple but different copies of the 16S rRNA gene in species such as Nocardia nova (7, 9) and/or the presence of intraspecies 16S rRNA gene sequence polymorphisms (or “sequence types” [STs]) in N. nova, Nocardia cyriacigeorgica, and other species (14, 21).As such, the continuing evaluation of alternate gene targets to facilitate species identification is important. Sequence polymorphisms within the Nocardia 65-kDa heat shock protein (hsp65), essential secretory protein SecA1 (secA1), gyrase B (gyrB), and 16S-23S rRNA intergenic spacer (ITS) region genes have been reported to enable species level identification (10, 18, 22-24). In particular, sequence variability within a portion (470 bp) of the secA1 gene locus (in conjunction with analysis of deduced amino acid sequences of the SecA1 protein) has shown promise in recognizing and discriminating between the major Nocardia spp. (10). However, data on the application of secA1 gene sequencing in the clinical microbiology laboratory for the identification of Nocardia isolates are few. In one report, reference (n = 30 species), and clinical Nocardia isolates were correctly identified by secA1 gene sequencing (10); in the only other published study, this approach assisted with identification of a novel Nocardia species from soil (16). Evaluation of larger numbers of clinical isolates is essential for establishing a robust repository of secA1 gene sequences.Our laboratory, which provides regional microbiology services to a large number of health care institutions, has undertaken routine species identification by partial (5′-end 606-bp) 16S rRNA gene sequencing of Nocardia isolates since 2005. In the course of evaluating this approach to providing species identification, we identified significant intraspecies sequence heterogeneity within certain species, such as N. nova and Nocardia brasiliensis (14), highlighting the need to recognize species-specific sequence-based genetic types, or sequence types. Here, to explore the potential of sequence analysis of the secA1 gene as an adjunct to, or a possible substitute for, 16S rRNA gene sequencing, we performed species identification of 120 Nocardia reference and clinical isolates representing the 16 most clinically relevant species by secA1 gene sequence analysis and compared the results with 5′-end 606-bp 16S rRNA gene sequencing. We also report on the genetic diversity of the Nocardia secA1 gene.     

Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates

Mixed infections and heteroresistance of Mycobacterium tuberculosis contribute to the difficulty of diagnosis, treatment, and control of tuberculosis. However, there is still no proper solution for these issues. This study aimed to investigate the potential relationship between mixed infections and heteroresistance and to determine the high-risk groups related to these factors. A total of 499 resistant and susceptible isolates were subjected to spoligotyping and 24-locus variable-number tandem repeat methods to analyze their genotypic lineages and the occurrence of mixed infections. Two hundred ninety-two randomly selected isolates were sequenced on their rpoB gene to examine mutations and heteroresistance. The results showed that 12 patients had mixed infections, and the corresponding isolates belonged to Manu2 (n = 8), Beijing (n = 2), T (n = 1), and unknown (n = 1) lineages. Manu2 was found to be significantly associated with mixed infections (odds ratio, 47.72; confidence interval, 9.68 to 235.23; P < 0.01). Four isolates (1.37%) were confirmed to be heteroresistant, which was caused by mixed infections in three (75%) isolates; these belonged to Manu2. Additionally, 3.8% of the rifampin-resistant isolates showing no mutation in the rpoB gene were significantly associated with mixed infections (χ², 56.78; P < 0.01). This study revealed for the first time that Manu2 was the predominant group in the cases of mixed infections, and this might be the main reason for heteroresistance and a possible mechanism for isolates without any mutation in the rpoB gene to become rifampin resistant. Further studies should focus on this lineage to clarify its relevance to mixed infections.     

DisseminatedNocardia farcinica infection in an AIDS patient

This report describes an AIDS patient presenting with disseminatedNocardia farcinica infection diagnosed by percutaneous kidney biopsy. The isolate was initially identified asNocardia asteroides. ThoughNocardia asteroides remains sensitive to most antimicrobial agents,Nocardia farcinica is resistant to gentamicin, tobramycin and cephalosporins and is indistinguishable fromNocardia asteroides by regular laboratory methods. In view of the rising incidence of infections withNocardia farcinica, third-generation cephalosporins should not be used in the initial management ofNocardia infections, and all isolates should be submitted for antibiotic susceptibility testing.     

Streptococcus pneumoniae ocular infections,prominent role of unencapsulated isolates in conjunctivitis

The aim of this study was to determine the characteristics and shifts in serotype distribution of pneumococcal isolates causing ocular infections in a region of northern Spain in two periods: 1999–2010 for episodes of conjunctivitis (n = 612) and 1980–2010 for uncommon and more severe non-conjunctival ocular infections (n = 36). All isolates were serotyped and non-typeable isolates were confirmed as unencapsulated by multiplex-PCR of the lytA, ply and cpsA genes. Genotyping was done by pulsed-field gel electrophoresis and multi-locus sequence typing. Most conjunctivitis cases occurred in children under 5 years old (89.5%), and more severe non-conjunctival ocular infections occurred in patients older than 25 years (86.1%). Unencapsulated isolates were detected in 213 conjunctivitis episodes (34.8%) and one non-conjunctival infection (2.8%). Rates of unencapsulated isolates were similar throughout the study. Among 399 conjunctival encapsulated isolates, the most prevalent were serotypes 19A (n = 53), 15B (n = 30), 6A (n = 27), 19F (n = 25), 23F (n = 21) and 6B (n = 17). The most prevalent serotypes in non-conjunctival infections were serotype 3 (n = 4), 23F (n = 4), 6B (n = 3) and 19A (n = 3). Conjunctivitis caused by serotypes included in the hepta-valent pneumococcal conjugate vaccine steadily decreased, accounting for 34.9% (22/63) in 1999–2001, 19.7% (23/117) in 2002–04, 13.6% (33/242) in 2005–07 and 3.2% (6/190) in 2008–10. Among the 213 unencapsulated isolates, 31 different pulsed-field gel electrophoresis patterns were identified. The main clonal complexes (CC) were CC941 (ST941, ST942), CC448 (ST448) and CC344 (ST344, ST3097). CC941 was the predominant CC in 1999–2001, 2002–04 and 2005–07, being replaced by CC448 in 2008–10. The multidrug-resistant CC344 was present throughout the study.     

Pulmonary nocardiosis: Risk factors and species distribution from a high burden centre

Nocardiosis is a clinical and diagnostic challenge. This was a retrospective study carried out on cases of pulmonary nocardiosis presenting over 15 years.Clinical data was retrieved using the electronic patient records. Vitek MS 3.2 (MALDI TOF MS) was carried out on 22 isolates and sequencing on another 9 isolates.Of 71 patients presenting with pulmonary nocardiosis, 58 (81.6%) were on immunosuppressant therapy, 26 (46%) had a previous lung pathology, 11 (8%) were HIV associated. Disseminated disease was seen in 6 (8.4%). There were 8 (11.26%) deaths in this cohort of patients. Of 31/71 identified to species, the most common were Nocardia cyriacigeorgica (n ?= ?11) followed by Nocardia farcinica (n ?= ?9).     

Clinical and microbiological characteristics of infections caused by various Nocardia species in Taiwan: a multicenter study from 1998 to 2010

This multicenter study in Taiwan investigated the clinical presentations of various Nocardia species infections based on 16S rRNA sequence analysis. Patients with nocardiosis in four large medical centers from 1998 to 2010 were included. A total of 100 preserved nonduplicate isolates causing human infection were identified as Nocardia species. Sequencing analysis of 16S rRNA confirmed that 35 of 36 N. asteroides isolates identified by conventional tests were non-asteroides Nocardia species, and that two of 50 N. brasiliensis isolates had also been initially misidentified. N. brasiliensis (50%) was the most common pathogen, followed by N. cyriacigeorgica (18%). In addition, several rare pathogens were identified, including N. asiatica, N. rhamnosiphila, N. abscessus, N. transvalensis, N. elegans, and N. carnea. Primary cutaneous infection was the most common presentation, noted in 55 (55%) patients, while pulmonary infection presented in 26 (26%) patients. The crude mortality rate was 6.7% (6/89), and was lowest for primary cutaneous infection (2.2%) and highest for disseminated disease and pulmonary infection (16.7%). In conclusion, N. brasiliensis and N. cyriacigeorgica were the most common pathogens causing nocardiosis in Taiwan. Molecular methods for identifying Nocardia to the species level are mandatory for better understanding the epidemiology and clinical characteristics of patients with nocardiosis.     

Pathogenicity and Phenotypic Characterization of Enterotoxigenic Escherichia coli Isolates from a Birth Cohort of Children in Rural Egypt

Enterotoxigenic Escherichia coli (ETEC) has consistently been the predominant bacterial cause of diarrhea in many birth cohort- and hospital-based studies conducted in Egypt. We evaluated the pathogenicity of ETEC isolates in a birth cohort of children living in a rural community in Egypt. Between 2004 and 2007, we enrolled and followed 348 children starting at birth until their second year of life. A stool sample and two rectal swabs were collected from children during twice-weekly visits when they presented with diarrhea and were collected every 2 weeks if no diarrhea was reported. From routine stool cultures, five E. coli-like colonies were screened for ETEC enterotoxins using a GM1 enzyme-linked immunosorbent assay (ELISA). The isolates were screened against a panel of 12 colonization factor antigens (CFAs) by a dot blot assay. A nested case-control study evaluated the association between initial or repeat excretion of ETEC and the occurrences of diarrhea. The pathogenicity of ETEC was estimated in symptomatic children compared to that in asymptomatic controls. ETEC was significantly associated with diarrhea (crude odds ratio, 1.37; 95% confidence interval [CI], 1.24 to 1.52). The distribution of ETEC enterotoxins varied between the symptomatic children (44.2% heat-labile toxin [LT], 38.5% heat-stable toxin [ST], and 17.3% LT/ST) and asymptomatic children (55.5% LT, 34.6% ST, and 9.9% LT/ST) (P < 0.001). The CFAs CFA/I (n = 61), CS3 (n = 8), CS1 plus CS3 (n = 24), CS2 plus CS3 (n = 18), CS6 (n = 45), CS5 plus CS6 (n = 11), CS7 (n = 25), and CS14 (n = 32) were frequently detected in symptomatic children, while CS6 (n = 66), CS12 (n = 51), CFA/I (n = 43), and CS14 (n = 20) were detected at higher frequencies among asymptomatic children. While all toxin phenotypes were associated with diarrheal disease after the initial exposure, only ST and LT/ST-expressing ETEC isolates (P < 0.0001) were associated with disease in repeat infections. The role of enterotoxins and pathogenicity during repeat ETEC infections appears to be variable and dependent on the coexpression of specific CFAs.     

Antimicrobial Susceptibility of Leptospira spp. Isolated from Environmental,Human and Animal Sources in Malaysia

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes of the genus Leptospira. The aim of this study was to evaluate the susceptibility of isolates obtained from different hosts. A total of 65 Leptospira isolates from humans (n = 1), zoonoses (rat, n = 60; dog, n = 1; swine, n = 1) and environment (n = 2) were tested against six antibiotics. All the isolates were resistant to trimethoprim and sulphamethoxazole and had high MIC toward chloramphenicol (MIC₉₀: 6.25 μg/ml). All except one environment isolate were sensitive to ampicillin, doxycycline and penicillin G.     

Multicenter Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Study for Identification of Clinically Relevant Nocardia spp.

This multicenter study analyzed Nocardia spp., including extraction, spectral acquisition, Bruker matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification, and score interpretation, using three Nocardia libraries, the Bruker, National Institutes of Health (NIH), and The Ohio State University (OSU) libraries, and compared the results obtained by each center. A standardized study protocol, 150 Nocardia isolates, and NIH and OSU Nocardia MALDI-TOF MS libraries were distributed to three centers. Following standardized culture, extraction, and MALDI-TOF MS analysis, isolates were identified using score cutoffs of ≥2.0 for species/species complex-level identification and ≥1.8 for genus-level identification. Isolates yielding a score of <2.0 underwent a single repeat extraction and analysis. The overall score range for all centers was 1.3 to 2.7 (average, 2.2 ± 0.3), with common species generally producing higher average scores than less common ones. Score categorization and isolate identification demonstrated 86% agreement between centers; 118 of 150 isolates were correctly identified to the species/species complex level by all centers. Nine strains (6.0%) were not identified by any center, and six (4.0%) of these were uncommon species with limited library representation. A categorical score discrepancy among centers occurred for 21 isolates (14.0%). There was an overall benefit of 21.2% from repeat extraction of low-scoring isolates and a center-dependent benefit for duplicate spotting (range, 2 to 8.7%). Finally, supplementation of the Bruker Nocardia MALDI-TOF MS library with both the OSU and NIH libraries increased the genus-level and species-level identification by 18.2% and 36.9%, respectively. Overall, this study demonstrates the ability of diverse clinical microbiology laboratories to utilize MALDI-TOF MS for the rapid identification of clinically relevant Nocardia spp. and to implement MALDI-TOF MS libraries developed by single laboratories across institutions.     

Infection Caused by Nocardia farcinica: Case Report and Review

 Nocardia farcinica is a rare Nocardia species causing localised and disseminated infections. A case of Nocardia farcinica infection is presented, and 52 cases previously reported in the literature are reviewed. The hosts usually had predisposing conditions (85%), and acquired the infection through the respiratory tract or skin; the infection then often spread to the brain, kidney, joints, bones and eyes. Pulmonary or pleural infections (43%), brain abscesses (30%) and wound infections (15%) which failed to respond to conventional antimicrobial therapy were the more frequent forms of infection. Nocardia farcinica was frequently isolated from pus (100% of samples), bronchial secretions (41%) and biopsy specimens (63%), but isolation from blood and urine, as in the case presented here, is rare. Antibiotic therapy was adequate in 61% of the patients in whom it was specified, the agents most frequently given being trimethoprim-sulfamethoxazole (54%), amikacin combined with imipenem (7%) and amoxicillin-clavulanate (7%). The high mortality (31%) can be attributed to the severe underlying diseases present, difficulties encountered in identifying the pathogen, inappropriate therapy and late initiation of therapy. Although an infrequent pathogen, Nocardia farcinica should be kept in mind as a cause of infection especially in immunosuppressed patients with indolent infections not responding to third-generation cephalosporins.     

Capsular serotypes and multilocus sequence types of bacteremic Klebsiella pneumoniae isolates associated with different types of infections

We investigated the epidemiology of different serotypes of Klebsiella pneumoniae isolates causing bacteremic liver abscess (LA) using multilocus sequence typing (MLST). MLST and molecular typing were performed for 41 K1 (19 LA), 37 K2 (5 LA), and 33 non-K1/K2 (6 LA) isolates that were derived from a previous one-year K. pneumoniae bacteremia cohort. Capsular serotypes and rmpA of these isolates were determined by polymerase chain reaction (PCR) methods. Among the 41 K1 isolates, 39 were ST23 and the remaining two isolates were ST23 single-locus variant. There were 11 STs among K2 isolates. ST65 was the most common (n?=?10), followed by ST86, ST373, and ST375. Only ST65 (n?=?3), ST373 (n?=?1), and ST375 (n?=?1) caused LA, and ST65 was a three-locus variant of ST23. For non-K1/K2 isolates, the ST types varied widely. ST218 (K57) was the most common type (n?=?6, 18 %), and it was a single-locus variant of ST23 and caused two cases of LA. The existences of rmpA among serotypes varied (100 % for K1, 89 % for K2, and 55 % for non-K1/K2). For isolates causing LA, all of them were positive for rmpA. For non-K1/K2 isolates causing infections other than LA, the positivity of rmpA ranged from 0 % (biliary tree infection) to 67 % (pneumonia). In this one-year cohort, all K1 isolates were ST23 or its single-locus variants, but the composition of ST types among K2 isolates was quite variable. ST23 and its one- (ST1005 and ST218) and three-locus (ST65) variants comprised 80 % of isolates causing LA.     

In vitro activity of fluoroquinolones against clinical isolates of Nocardia identified by partial 16S rRNA sequencing

Fluoroquinolones have several properties that make them potentially attractive candidates for the treatment of Nocardia infections, but information regarding their in vitro activity is limited. Minimum inhibitory concentrations (MIC) of five fluoroquinolones and other antimicrobials were determined by the reference broth dilution and E-test methods for 33 consecutive clinical isolates of Nocardia speciated by 16S rRNA gene sequences. The isolates included: Nocardia cyriacigeorgica (n = 6), N. nova (n = 8), N. farcinica (n = 8), N. brasiliensis (n = 3), N. asteroides (n = 4), and N. veterana (n = 4). MIC₅₀/MIC₉₀ results for ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin by broth dilution were 32/32, 2/4, 1/4, 32/32, and 2/2 μg/ml, respectively. The MICs by broth dilution and E-test were within a two-fold doubling dilution for 94%, 97%, 97%, 100%, and 100% of isolates for ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, respectively. For ciprofloxacin, the E-test results showed either complete categorical agreement or minor error compared to the reference broth dilution method for 97% (32/33) of the isolates. For other fluoroquinolones, using Streptococcus pneumoniae breakpoints, 94% (124/132) of MIC results by E-test showed either complete agreement or minor error compared to the reference broth dilution method. Fluoroquinolones show variable in vitro activity against clinical isolates of Nocardia spp., and MICs determined by the E-test show reasonable agreement with those determined by the reference broth dilution method.     

Microbiological and clinical features of nine cases with nocardial infections

Following recent advance in medical technology, the increase of immunocompromised patients results in an increase of opportunistic infections such as nocardiosis. However, little is known about relationships between clinical features of nocardial infections and each Nocardia species, especially newly identified ones. Therefore, we identified clinical isolates of Nocardia species by genetic methods and analyzed clinical features of nocardiosis. Nine clinical isolates were obtained in Kyushu University Hospital from 2005 to 2008. Six different Nocardia species were identified by 16Sr RNA: Nocardiafarcinia (n=2), Nocardia brasiliensis (n=2), Nocardia cyriacigeorgica (n=2), Nocardia transvalensis (n=1), Nocardia araoensis (n=1) and Nocardia testacea (n=1). The underlying diseases of 9 patients were pulmonary diseases(n=5), malignant diseases(n=3), collagen diseases(n=1) or primary immunodeficiency diseases (n=l). According to antimicrobial susceptibility testing, none of them was resistant to minocycline or linezolid. Among seven isolates from respiratory specimens, one was resistant to imipenem, sulfamethoxazole/trimethoprim and amikacin, two were to ciprofloxacin. Three species identified recently (N cyriacigeorgica, N. araoensis and N. testacea) were involved in this study and most of them were considered as infectious pathogens to human. These data suggested the identification of Nocardia to the species level and susceptibility testing were important for diagnosis as infectious diseases and treatments.     

Allocation of Klebsiella pneumoniae Bloodstream Isolates into Four Distinct Groups by ompK36 Typing in a Taiwanese University Hospital

The OmpK36 porin plays a role in carbapenem resistance and may contribute to bacterial virulence in Klebsiella pneumoniae. This study aimed to investigate the characteristics of different groups of K. pneumoniae separated by ompK36 typing. Among 226 nonduplicate K. pneumoniae bloodstream isolates collected at a Taiwanese hospital in 2011, four ompK36 types, designated types A, B, C, and D, were identified by PCR in 61, 28, 100, and 36 isolates, respectively; 1 isolate was untypeable. Statistical analysis showed significantly higher rates of antimicrobial resistance (all tested antibiotics except meropenem), extended-spectrum β-lactamases or DHA-1 (47.5% together), Qnr-type quinolone resistance determinants (50.8%), and IncFIIA-type plasmids (49.2%) in group A than in others. Seventeen isolates were identified as belonging to 3 international high-risk clones (4 sequence type 11 [ST11], 10 ST15, and 3 ST147 isolates); all isolates but 1 ST15 isolate were classified in group A. The significant characteristics of group C were hypermucoviscosity (62.0%) and a higher virulence gene content. This group included all serotype K1 (n = 30), K2 (n = 25), and K5 (n = 3) isolates, 6 of 7 K57 isolates, all isolates of major clones associated with pyogenic liver abscesses (29 ST23, 11 ST65, 5 ST86, 7 ST373, and 1 ST375 isolates), and 16 (94.1%) of 17 isolates causing bacteremic liver abscesses. Twelve (42.9%) of the group B isolates were responsible for bacteremic biliary tract infections. Group D was predominant (83.3%) among 12 K20 isolates. This study suggests that most clinical K. pneumoniae isolates can be allocated into four groups with distinct characteristics based on ompK36 types.     

Molecular epidemiology of Acinetobacter baumannii and Acinetobacter nosocomialis in Germany over a 5-year period (2005–2009)

To investigate the species distribution within the Acinetobacter calcoaceticus–Acinetobacter baumannii complex and the molecular epidemiology of A. baumannii and Acinetobacter nosocomialis, 376 Acinetobacter isolates were collected prospectively from hospitalized patients at 15 medical centres in Germany during three surveillance studies conducted over a 5-year period. Species identification was performed by molecular methods. Imipenem minimum inhibitory concentrations (MIC) were determined by broth microdilution. The prevalence of the most common carbapenemase-encoding genes was investigated by oxacillinase (OXA) -multiplex polymerase chain reaction (PCR). The molecular epidemiology was investigated by repetitive sequence-based PCR (rep-PCR; DiversiLab?). Acinetobacter pittii was the most prevalent Acinetobacter species (n = 193), followed by A. baumannii (n = 140), A. calcoaceticus (n = 10) and A. nosocomialis (n = 8). The majority of A. baumannii was represented by sporadic isolates (n = 70, 50%) that showed unique rep-PCR patterns, 25 isolates (18%) clustered with one or two other isolates, and only 45 isolates (32%) belonged to one of the previously described international clonal lineages. The most prevalent clonal lineage was international clone (IC) 2 (n = 34) and IC 1 (n = 6). According to CLSI, 25 A. baumannii isolates were non-susceptible to imipenem (MIC ≥ 8 mg/L), all of which produced an OXA-58-like or OXA-23-like carbapenemase. The rate of imipenem susceptibility among A. baumannii isolates decreased from 96% in 2005 to 76% in 2009. All other Acinetobacter isolates were susceptible to imipenem. The population structure of carbapenem-susceptible A. baumannii in Germany is highly diverse. Imipenem non-susceptibility was strongly associated with the clonal lineages IC 2 and IC 1. These data underscore the high clonality of carbapenem-resistant A. baumannii isolates.