- Volume 58, Issue 2, 2009

We screened 500 pregnant women who had no risk factors for Streptococcus agalactiae vaginal carriage, and isolated 39 S. agalactiae strains (8 %). The density of carriage was low in 16 cases (41 %), intermediate in 16 cases (41 %) and heavy in seven cases (18 %). Strains were mostly of serotype III (41 %), Ia (26 %) and V (18 %). Thirty-five strains had at least one of five genetic markers that have been associated with virulent phylogenetic subgroups of strains. Using PCR, nine strains (23 %) were identified as belonging to CC17. The 39 vaginal strains that were studied exhibited a substantial genetic diversity; there were 39 PFGE profiles and 13 variants defined on the basis of the five genetic markers studied. The prevalence of the studied genetic characteristics was similar for strains associated with all three classes of density of carriage. These data suggest that genetic features that are markers of S. agalactiae strains able to invade the central nervous system of neonates are not determinants for vaginal adaptation.

The genetic heterogeneity and antifungal susceptibility patterns of Candida parapsilosis isolated from blood cultures of patients were investigated in this study. Randomly amplified polymorphic DNA (RAPD) analysis generated 5 unique profiles from 42 isolates. Based on the major DNA fragments of the RAPD profiles, the isolates were identified as RAPD type P1 (29 isolates), P2 (6 isolates), P3 (4 isolates), P4 (2 isolates) and P5 (1 isolate). Sequence analysis of the internal transcribed spacer (ITS) gene of the isolates identified RAPD type P1 as C. parapsilosis, P2 and P3 as Candida orthopsilosis, P4 as Candida metapsilosis, and P5 as Lodderomyces elongisporus. Nucleotide variations in ITS gene sequences of C. orthopsilosis and C. metapsilosis were detected. Antifungal susceptibility testing using Etests showed that all isolates tested in this study were susceptible to amphotericin B, fluconazole, ketoconazole, itraconazole and voriconazole. C. parapsilosis isolates exhibited higher MIC50 values than those of C. orthopsilosis for all of the drugs tested in this study; however, no significant difference in the MICs for these two Candida species was observed. The fact that C. orthopsilosis and C. metapsilosis were responsible for 23.8 and 4.8 % of the cases attributed to C. parapsilosis bloodstream infections, respectively, indicates the clinical relevance of these newly described yeasts. Further investigations of the ecological niche, mode of transmission and virulence of these species are thus essential.

The prevalence of Aspergillus endocarditis (AE) is increasing in the hospital population. Aspergillus species contribute to approximately 25 % of all cases of fungal endocarditis. This study is a descriptive report of the use of nested PCR to detect DNA specific for Aspergillus species in serum for the diagnosis of cardiac infections. Open heart surgery was performed on patients and collected samples were examined microscopically and cultured. Ten sera in total from five patients were extracted for Aspergillus DNA and nested PCR with Aspergillus species primers was carried out. The lowest limit of detection for the PCR assay was 1 c.f.u. (ml serum) −1. The PCR was positive in three patients. Culture of valvular tissue confirmed the growth of Aspergillus fumigatus in one patient and Aspergillus niger in two patients. In this study we have demonstrated the presence of invasive aspergillosis in patients who had undergone open heart surgery and the usefulness of a molecular assay for the diagnosis of AE.

A 30 month prospective study of Acinetobacter species encountered in the Central Pathology Laboratory of St James's Hospital, Dublin, Ireland, was conducted to investigate the prevalence and molecular epidemiology of carbapenem resistance in such isolates. Acinetobacter genomic species 3 (AG3) was found to be the predominant Acinetobacter species (45/114, 39 %) in our institution. A total of 11 % of all Acinetobacter species (12/114) and 22 % of AG3 isolates (10/45) were carbapenem resistant. Carbapenem resistance was mediated by Ambler class D β-lactamase OXA-23 in all 12 isolates, with insertion sequence ISAba1 found upstream of bla OXA-23. ISAba1 was also found upstream of bla ADC-25, which encodes the enzyme AmpC, in an Acinetobacter baumannii isolate, and upstream of the aminoglycoside-acetyltransferase-encoding gene aacC2 in three AG3 isolates. Inter-species plasmidic transfer was most likely involved in the emergence and spread of bla OXA-23 among the Acinetobacter isolates within our institution. The emergence of carbapenem resistance was associated not only with prior carbapenem use but also with the use of other antimicrobial agents, most notably β-lactam/β-lactamase-inhibitor combinations. The study demonstrated the emerging trend of carbapenem resistance in the wider context of the Acinetobacter genus, and reiterated the paramount importance of the prudent use of antimicrobial agents, stringent infection control measures and resistance surveillance of pathogens.

The emergence of carbapenem-hydrolysing metallo-β-lactamases (MBLs) is a serious threat to the clinical utility of carbapenems. This study identified plasmid- and integron-borne bla IMP-1 and bla IMP-10 in clinical isolates of Serratia marcescens. The bla IMP-1 and bla IMP-10 gene cassettes were carried by a class 1 integron and followed by the aac(6′)-IIc gene cassette. The bla IMP-1 and bla IMP-10 gene cassettes were preceded by a weak Pant promoter, TGGACA(N)17TAAGCT, and an inactive P2 promoter, TTGTTA(N)14TACAGT. These genes were easily transferred to Escherichia coli by conjugation and transformation, indicating that they are located on transferable plasmids. Due to the acquisition of bla IMP-1, the susceptibility of E. coli transconjugants to imipenem, meropenem, panipenem and biapenem decreased by 32-, 256-, 64- and 128-fold, respectively. In comparison, after gaining bla IMP-10, the susceptibility of E. coli transconjugants to the four carbapenems decreased by 64-, 2048-, 256- and 64-fold, respectively. Strains harbouring bla IMP-10 showed higher-level resistance to imipenem, meropenem and panipenem than the strains harbouring bla IMP-1, although the nucleotide sequences of the class 1 integrons carrying bla IMP-10 and bla IMP-1 were identical except for a single point mutation.

Members of the Streptococcus anginosus group (SAG) are frequently involved in pyogenic infections in humans. In the present study, the antimicrobial susceptibility of 141 clinical SAG isolates to six antimicrobial agents was analysed by agar dilution. All isolates were susceptible to penicillin, cefotaxime and vancomycin. However, 12.8 % displayed increased MIC values (0.12 mg l−1) for penicillin. Resistance to erythromycin was detected in eight (5.7 %) isolates. Characterization of the erythromycin-resistant isolates with the double-disc diffusion test revealed Macrolide-Lincosamide-StreptograminB and M-type resistance in six and two isolates, respectively. The erythromycin-resistant isolates were further characterized by PCR for the resistance genes ermA, ermB and mefA. Resistance and intermediate resistance to ciprofloxacin were detected in two and six isolates, respectively. Molecular typing by PFGE revealed a high genetic heterogeneity among the SAG isolates and no evidence for a clonal relationship between the erythromycin-resistant isolates. Our data show that resistance to erythromycin, clindamycin and ciprofloxacin has emerged among SAG isolates in Germany. The implications of these findings for susceptibility testing and antimicrobial therapy of SAG infections are discussed.

A total of 32 Vibrio cholerae isolates were collected during a recent large cholera outbreak in Eastern India. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. Two multiplex PCR assays confirmed the presence of various toxigenic and pathogenic genes – ace, ctxAB, hlyA, ompU, ompW, rfbO1, rtx, tcp, toxR and zot – in all of the isolates. Sequencing of the ctxB gene from the isolates revealed a novel mutation in the gene. Sequencing also confirmed the presence of altered cholera toxin B of the classical biotype in all of the El Tor isolates, suggesting infection of isolates by classical CTXΦ. The molecular diversity of V. cholerae isolates studied by enterobacterial repetitive intergenic consensus sequence PCR, BOX-PCR and randomly amplified polymorphic DNA analysis uniformly showed the clonal relationship among the outbreak V. cholerae O1 isolates. The results of this study suggest that cholera-causing V. cholerae strains are constantly evolving in epidemic areas, highlighting the potential of the emergence of more virulent strains.

Retrospective analysis led to the detection of two Vibrio cholerae variant O1 strains (VC51 and VC53), which were isolated in 1992 in Kolkata from clinical cases, with identical traits to 2004 Mozambique variant O1 strains. The Mozambique O1 strains that caused a huge outbreak in 2004 have been shown to have phenotypic traits of both classical and El Tor biotypes, and thereby have been reported as variant. Our study demonstrated that two O1 strains isolated in Kolkata during 1992 were of the El Tor background as evidenced by polymyxin B (50 U ml−1) resistance, positivity in Voges–Proskauer reactions and sensitivity to biotype-specific vibrio phages. With the features of classical CTX prophage, localization in the small chromosome, and an absence of RS1 and pTLC, both Mozambique and Kolkata strains appeared to be identical. Furthermore, two Kolkata strains exhibited an identical ribotype to that of the Mozambique variant, displaying ribotype pattern RI that had been assigned to Kolkata V. cholerae O1 strains isolated on or before 1992. NotI pulsotype analysis indicated that these 1992 Kolkata strains along with the Mozambique variant O1 belonged to very closely related clones. Considering the chronological events, and the typical identity at the phenotypic and the genotypic level between the two O1 strains isolated during 1992 from Kolkata and during 2004 from Mozambique, we propose that some of the 1992 Kolkata O1 strains might have acted as progenitors for Mozambique variant O1 strains.

Brachyspira pilosicoli is an anaerobic spirochaete that colonizes the large intestine of humans and various species of animals and birds. The spirochaete is an important enteric pathogen of pigs and poultry, but its pathogenic potential in humans is less clear. In the current study, the occurrence of B. pilosicoli in faecal samples from 766 individuals in two different population groups in Perth, Western Australia, was investigated by selective anaerobic culture. Of 586 individuals who were long-term residents of Perth, including children, elderly patients in care and in hospital and individuals with gastrointestinal disease, only one was culture positive. This person had a history of diverticulitis. In comparison, faeces from 17 of 180 (9.4 %) Indonesians who were short- or medium-term visitors to Perth were positive for B. pilosicoli. The culture-positive individuals had been in the city for between 10 days and 4.5 years (median 5 months). Resampling of subsets of the Indonesians indicated that all negative people remained negative and that some positive individuals remained positive after 5 months. Two individuals had pairs of isolates recovered after 4 and 5 months that had the same PFGE types, whilst another individual had isolates with two different PFGE types that were identified 2 months apart. Individuals who were culture-positive were likely to have been either colonized in Indonesia before arriving in Perth or infected in Perth following contact with other culture-positive Indonesians with whom they socialized. Colonization with B. pilosicoli was not significantly associated with clinical signs at the time the individuals were tested, although faeces with wet-clay consistency were 1.5 times more likely (confidence interval 0.55–4.6) than normal faeces to contain B. pilosicoli.

The Mycobacterium avium complex (MAC) is the most frequently isolated species among non-tuberculous mycobacteria (NTM) clinical isolates. Physicians pay attention to the differential diagnosis of the disease caused by MAC from tuberculosis because of their similar clinical presentations. Expression of the macrophage-induced gene (mig) is one of the virulence phenotypes in MAC, but it has not been determined whether the presence of the mig gene itself has any relationship with clinical disease or whether it is merely a marker for MAC. To uncover the significance of the mig gene among MAC clinical isolates, positive cultures from respiratory specimens from patients in a tertiary referral centre were identified by sequencing the 16S rRNA gene. The mig gene was also evaluated using PCR and sequence analysis. The medical records from the patients were reviewed retrospectively. The diagnostic criteria from the American Thoracic Association were adopted for the diagnosis of NTM lung disease. A total of 45 MAC clinical isolates were identified over a period of 1 year. Following 16S rRNA sequencing, all of the 23 M. avium isolates were categorized as sequevar I. Among the 22 Mycobacterium intracellulare isolates, 18 strains were identified as M. intracellulare sequevar I and the remaining four consisted of one each of sequevars II, III, IV and V. The proportion of cases that fitted the diagnostic criteria of NTM lung disease was 26.7 % (12/45). The mig PCR results were 100 % positive for the MAC isolates studied, irrespective of their species, sequevar or disease-causing properties. However, following bootstrap analysis of the mig sequences, we observed definite grouping between M. avium and M. intracellulare. Thus the mig gene is a species-specific marker with distinct sequence diversity between the two species M. avium and M. intracellulare, but there is poor correlation between disease-causing properties and specific mig sequences.

This study was performed to assess the prevalence and genotypes of plasmid-borne extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in Escherichia coli in Korea. A total of 576 isolates of E. coli was collected from 12 Korean hospitals during May and July 2007. A phenotypic confirmatory test detected ESBLs in 82 (14.2 %) of the 576 E. coli isolates. The most common types of ESBLs identified were CTX-M-14 (n=32) and CTX-M-15 (n=27). The prevalence and diversity of the CTX-M mutants, including CTX-M-15, CTX-M-27 and CTX-M-57, with significant hydrolytic activity against ceftazidime were increased. PCR experiments detected genes encoding plasmid-borne AmpC β-lactamases in 15/56 cefoxitin-intermediate or cefoxitin-resistant isolates, and the most common type of AmpC β-lactamase identified was DHA-1 (n=10). These data suggest that the incidence of ESBLs in E. coli has increased as a result of the dissemination of CTX-M enzymes in Korea. In addition, CTX-M-22, CTX-M-27 and CTX-M-57 have appeared in Korea.

Staphylococcus pettenkoferi is a recently isolated human pathogen with only a few reported cases of infection. We report a case of bloodstream infection caused by S. pettenkoferi in a patient with pulmonary tuberculosis.