- Volume 59, Issue 9, 2010

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6×His were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

To determine the prevalence of cervical Chlamydia trachomatis genotypes in Iran for the first time and their association with three clinical symptoms/signs, i.e. abnormal vaginal discharge, lower abdominal pain (LAP) and swab-induced bleeding, and patient age, 620 cervical specimens were obtained from women with symptomatic genital infection referred to gynaecological clinics and 108 C. trachomatis-positive specimens were genotyped by direct omp1 gene PCR-RFLP analysis. Eight genotypes were identified. The most prevalent genotype was E (31.5 %), followed by F (23.1 %), D/Da (13 %), K (9.2 %), I (8.3 %), G (7.5 %), H (5.5 %) and J (1.9 %). For analysing the association of C. trachomatis genotypes with symptoms/signs and age, P-values were separately evaluated for genogroups and genotypes. The analysis of genogroups showed that women infected with genogroup F/G manifested the symptom of LAP significantly more often than those infected with the other genogroups (P=0.02), while the analysis of genotypes revealed that women infected with genotype F reported LAP slightly more often than women infected with the other genotypes (P=0.08). No significant correlation between genogroups and age was found; however, genotype E was somewhat less prevalent among women aged 25–34 years than among other age groups (P=0.08).

The measurement of IgG anti-pertussis toxin (IgG anti-PT) antibodies by ELISA is a frequently used method for studying the antibody responses after pertussis vaccination and after Bordetella pertussis infection. Such responses vary according to the different vaccines used as well as to the immunization and infection history of the participants. In the present study, the decay kinetics of the IgG anti-PT antibody response was determined for 71 Danish children and adults with bacteriologically confirmed B. pertussis infection and for 20 Danish adults booster-vaccinated with an acellular pertussis vaccine. For both groups, biphasic decay was seen, but the individual antibody responses varied greatly. No differences related to age were seen. Within each group, individual decay profiles showed parallel log-linear decay for the late part of the response. Antibody half-life was calculated for the late, slower part of the biphasic response curves for both groups (>5 months after diagnosis for individuals with confirmed infection; >3 months for vaccinated individuals). The median half-life for post-infection antibodies was 221 days [interquartile range (IQR) 159–314 days, 36 individuals], and the median half-life for post-vaccination antibodies was 508 days (IQR 428–616 days, 14 individuals). This difference was statistically significant (P<0.0001). Thus, in this setting, we found that the IgG anti-PT antibody decay after an infection with B. pertussis is more than twice as fast as the decay after booster vaccination with an acellular pertussis vaccine. Such knowledge of the IgG anti-PT decay kinetics is crucial for interpretation of serological data that will be used either for diagnosis or for epidemiological studies and surveillance of B. pertussis infections.

We investigated the molecular epidemiological features of 94 Mycobacterium intracellulare-related strains, isolated from Korean patients, using sequence analysis targeting 3 independent chronometer molecules, hsp65, the internal transcribed spacer 1 region and the 16S rRNA gene. By collective consideration of these three gene-based approaches, the 94 strains were divided into 5 groups (INT1, INT2, INT3, INT4 and INT5). The frequencies of genotype INT1, 2, 3, 4 and 5 in the 94 isolates were 57.4 % (54), 27.7 % (26), 6.4 % (6), 5.3 % (5) and 3.2 % (3), respectively. When correlations between genotypes and clinical parameters (age, sex, radiological type and the presence of a cavity) were analysed in 78 patients with non-tuberculous mycobacteria pulmonary diseases, no relationships were observed with respect to age, sex and radiological type, but genotype and the presence of a cavity tended to be related (P=0.051).

A total of 542 clinical isolates of aerobic Gram-negative bacilli from intra-abdominal infections were collected during 2008 from seven hospitals in India participating in the Study for Monitoring Antimicrobial Resistance Trends (SMART). Isolates were from various infection sources, the most common being gall bladder (30.1 %) and peritoneal fluid (31.5 %), and were mostly hospital-associated isolates (70.8 %) as compared to community-acquired (26.9 %). The most frequently isolated pathogens were Escherichia coli (62.7 %), Klebsiella pneumoniae (16.7 %) and Pseudomonas aeruginosa (5.3 %). Extended-spectrum β-lactamase (ESBL) rates in E. coli and K. pneumoniae were very high, at 67 % and 55 %, respectively. Most isolates exhibited resistance to one or more antibiotics. The most active drugs were generally ertapenem, imipenem and amikacin. However, hospital-acquired isolates in general, as well as ESBL-positive isolates, exhibited lower susceptibilities than community-acquired isolates. Further surveillance monitoring of intra-abdominal isolates from India is recommended.

The mtr gene complex in Neisseria meningitidis encodes an efflux pump that is responsible for export of antibacterial hydrophobic agents. The promoter region of the mtrCDE operon harbours an insertion sequence known as a Correia element, and a binding site for the integration host factor (IHF) is present at the centre of the Correia element. It has been suggested that the expression of the mtrCDE operon in meningococci is subject to transcriptional regulation by the IHF and post-transcriptional regulation by cleavage in the inverted repeat of the Correia element. The promoter region of the mtrCDE operon as well as the association of changes at that point with decreased susceptibility to antimicrobial drugs in 606 Neisseria meningitidis strains were analysed in this study. Two different deletions were present in the analysed region. The first one, found in seven strains, corresponded to absence of the Correia element. The second one, affecting the −10 region and first 100 bp of the mtrR gene and present in 57 isolates, was only found in ST-1624 isolates. None of the deletions were associated with decreased susceptibility to antimicrobial drugs. Although most of the meningococcal strains carry the Correia element at that position, its deletion is not an exception.

We studied the expression of DnaK and GroEL in Acinetobacter baumannii cells (strains ATCC 19606 and RS4) under stress caused by heat shock or antibiotics. A Western blot assay showed that DnaK and GroEL levels increased transiently more than 2-fold after exposure of bacterial cells to heat shock for 20 min at 50 °C. Heat induction of DnaK and GroEL was blocked completely when an inhibitor of transcription, rifampicin, was added 1 min before a temperature upshift to 50 °C, suggesting that the induction of these chaperones depends on transcription. A. baumannii cells pretreated at 45 °C for 30 min were better able to survive at 50 °C for 60 min than cells pretreated at 37 °C, indicating that A. baumannii is able to acquire thermotolerance. DnaK and GroEL were successfully induced in cells pre-incubated with a subinhibitory concentration of streptomycin. Moreover, bacterial cells pretreated for 30 min at 45 °C were better able to survive streptomycin exposure than cells pretreated at physiological temperatures. DnaK expression was upregulated in a multidrug-resistant strain of A. baumannii (RS4) in the presence of different antimicrobials (ampicillin+sulbactam, cefepime, meropenem and sulphamethoxazole+trimethoprim). This study is to the best of our knowledge the first to show that A. baumannii DnaK and GroEL could play an important role in the stress response induced by antibiotics.

This study was conducted to detect and analyse the presence of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae associated with a nosocomial outbreak at a Croatian hospital. During 2007, 162 K. pneumoniae isolates with reduced susceptibility to third-generation cephalosporins were collected from various hospital units and patient specimens. Most of the strains were isolated from urine (61 %), followed by blood cultures (13 %), wound swabs (13 %), tracheal aspirates (5 %), intra-abdominal abscess aspirates (4 %), intravascular catheters (3 %) and cerebrospinal fluid (1 %). Medical wards were the most important source of the isolates (46 %); 21 % of the isolates originated from surgical intensive-care units. All patients had infections acquired during their stay in hospital. No community-acquired infections were reported. Sixty of these isolates were chosen for further analysis. A double-disc synergy test (DDST) was used to detect ESBLs. MICs were determined by the broth microdilution method according to CLSI guidelines. The transferability of ceftazidime resistance was tested by conjugation (broth mating method). PCR was used to detect alleles encoding ESBL enzymes. Plasmids encoding ESBLs were extracted with the Macherey Nagel Mini kit according to the manufacturer's recommendations. The genotypes of the strains were compared by analysis of banding patterns generated by PFGE of XbaI-digested genomic DNA. ESBLs were found by DDST in all isolates. All strains were resistant to cefuroxime, ceftazidime, cefotaxime, ceftriaxone, aztreonam, piperacillin/tazobactam and ciprofloxacin. There was variable susceptibility/resistance to cefepime and gentamicin. No resistance to ceftazidime/clavulanate and carbapenems was observed. Only six strains transferred resistance to an Escherichia coli recipient strain, with low frequency. All isolates yielded an amplicon of 545 bp with consensus MA primers. Multiplex PCR was positive for group 1 CTX-M β-lactamases. Sequencing of selected amplicons revealed the presence of bla CTX-M-15, with coding regions containing identical nucleotide sequences. Similarly to isolates from India, our isolates contained the ISEcpI insertion sequence located upstream of the bla CTX-M-15 gene, which has recently been demonstrated to mobilize 3′-adjacent genes to transfer between DNA replicons. The isolates contained a large plasmid of approximately 150 kb. The isolates were assigned to five clusters (>85 % similarity), which contained subclusters. The results of this work provided insights into the molecular epidemiology of the spread of ESBLs in K. pneumoniae involved in an outbreak at a Croatian hospital. The hospital antibiotic policy resulted in ceftriaxone being the most heavily prescribed third-generation cephalosporin, which might be expected to select for cefotaximases such as CTX-M-15.

Staphylococcus aureus infections are a burden to healthcare systems. There remains a lack of understanding on the relative contributions of S. aureus infection in the healthcare and community settings. In this study, 59 S. aureus isolates were selected for molecular analysis. The mobile variant staphylococcal cassette chromosome mec type IV was present in both healthcare-associated meticillin-resistant S. aureus (HA-MRSA) and community-associated MRSA (CA-MRSA), as was the Panton–Valentine leukocidin gene. PFGE identified 24 distinct clonal groups whilst multi-locus sequence typing identified 26 different sequence types, including four with new combinations of alleles. This is the first time, to our knowledge, that a selection of CA and HA MSSA and MRSA strains have been subjected to molecular analysis and comparison in the UK. Definitions for CA-MRSA need further debate as the movement of strains between healthcare and community settings is confounding the use of epidemiological definitions.

The mouse disease model has the advantage of a broad array of immunological and genetic tools available for basic research. Some studies on transgenic and/or mutant mouse strains as models for experimental leptospirosis have been reported; however, the wider use of such models is hampered by a poor understanding of the outcome of experimental leptospiral infection among the different mouse strains available. Here, the outcome of infection by a virulent strain of Leptospira interrogans serogroup Icterohaemorrhagiae strain Cop was studied in four commonly used wild-type mouse strains: A, CBA, BALB/c and C57BL/6. The end points evaluated in this study were survival, presence of kidney lesions, leptospiral load in kidney samples, microscopic agglutination test titre and anti-leptospiral IgG antibody levels. As expected, none of the mouse strains were susceptible to lethal leptospirosis. However, these strains developed specific pathologies associated with sublethal leptospirosis. The A and C57BL/6 strains exhibited a high leptospiral load in kidney samples and the CBA and C57BL/6 strains developed severe inflammatory lesions, whilst the BALB/c strain proved to be the most resistant to subclinical leptospirosis.

The intracellular Gram-negative pathogen Francisella tularensis is the causative agent of tularaemia and is prevalent in many countries in the northern hemisphere. To determine whether the common marmoset (Callithrix jacchus) would be a suitable non-human primate model of inhalational tularaemia, a pathophysiology study was undertaken. Ten animals were challenged with ∼102 c.f.u. F. tularensis strain SCHU S4 (F. tularensis subsp. tularensis). To look for trends in the infection, pairs of animals were sacrificed at 24 h intervals between 0 and 96 h post-challenge and blood and organs were assessed for bacteriology, pathology and haematological and immunological parameters. The first indication of infection was a raised core temperature at 3 days post-challenge. This coincided with a number of other factors: a rapid increase in the number of bacteria isolated from all organs, more pronounced gross pathology and histopathology, and an increase in the immunological response. As the disease progressed, higher bacterial and cytokine levels were detected. More extensive pathology was observed, with multifocal lesions seen in the lungs, liver and spleen. Disease progression in the common marmoset appears to be consistent with human clinical and pathological features of tularaemia, indicating that this may be a suitable animal model for the investigation of novel medical interventions such as vaccines or therapeutics.

Here we report two cases of isolation of Aurantimonas altamirensis from pleural fluid and blood. The strains were identified by 16S rRNA gene sequencing. A. altamirensis appears to be a rare pathogen involved in unusual infectious processes, and must be isolated and studied at the molecular level for correct clinical diagnosis.