- Volume 59, Issue 7, 2010

Colonization factor antigens (CFAs) are important virulence factors in enterotoxigenic Escherichia coli (ETEC). Using a multiplex PCR and RT-PCR, this study tested the presence of common colonization factor-encoding genes and their expression in 50 ETEC strains isolated from stool specimens. The samples were from patients (children) with acute diarrhoea (cases) admitted to the Infectious Disease Hospital (Kolkata, India) and from normal children (controls) under 5 years of age from the community. The results indicated that coli surface antigen 6 (CS6) was the most prevalent CFA (78 %) expressed by these ETEC strains. Sequence analysis of both of the CS6 structural genes, i.e. cssA and cssB, in different ETEC isolates revealed the presence of point mutations in a systematic fashion. Based on the analysis of these variations, it was found that CssA had three alleles and CssB had two. Based on the allelic variations, subtyping of CS6 into AIBI, AIIBII, AIIIBI, AIBII and AIIIBII is proposed. The point mutations in the different alleles were reflected in a partial alteration in the secondary structure of both subunits, as determined by computational analysis. The functional significance of these changes was confirmed with cellular binding studies in Caco-2 cells with representative ETEC isolates. CS6 with AI or AIII allelic subtypes showed a higher binding capacity than AII, whereas BI showed stronger binding than BII. The AII and BII alleles were mostly detected in controls rather than in cases. The antibody specificity of BI and BII also varied due to alteration of the amino acids. Thus, CS6 variants are formed as a result of different allelic combinations of CssA and CssB, and these changes at the functional level might be important in the development of an effective ETEC vaccine.

Macrolide resistance in 169 Streptococcus agalactiae [group B streptococcus (GBS)] isolates originating from pregnant carriers was investigated. Using multiplex PCR the presence of genes encoding erythromycin resistance and capsular polysaccharides, as well as surface proteins, was determined. Random amplification of polymorphic DNA (RAPD) and PFGE were used to characterize specific clones among the isolates. In the examined population of women, erythromycin-resistant strains were found in 4.5 % of patients, whereas clindamycin-resistant strains were found in 3 % of patients, which was 16 % of strains resistant to erythromycin and 10 % of strains resistant to clindamycin among GBS isolates, respectively. Among the isolates, the largest percentage was represented by the constitutive macrolide–lincosamide–streptogramin B (cMLSB) phenotype (63 %), then the inductive macrolide–lincosamide–streptogramin B (iMLSB) phenotype (26 %) and the macrolide resistance (M) phenotype (11 %). The ermB gene was indicated in all isolates with the cMLSB phenotype and V serotype, whereas mefA/mefE genes were found in isolates with the M phenotype and Ia serotype. Among resistance isolates, serotype V was predominant (67 %), followed by serotypes II (15 %), Ia (11 %) and III (7 %). The most common surface protein encoding genes were alp3 (70 %), then rib (11 %), epsilon (7.5 %), bca (7.5 %) and alp2 (4 %). A statistically significant relationship between macrolide resistance, serotype V and the alp3 gene was demonstrated. PFGE, in comparison to the RAPD method, gave better genetic discrimination of GBS isolates. A relatively high genetic diversity among investigated strains was shown. In addition, the largest genetic homogeneity was found in serotype V.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced for bacterial identification. To our knowledge, this is the first study where the Biotyper 2.0 database (Bruker Daltonics) has been applied for bacterial identification in a local strain collection of molecularly defined Staphylococcus aureus. We showed that the accuracy of the Biotyper 2.0-based identification for 602 molecularly defined strains of S. aureus, irrespective of meticillin resistance, was equivalent to that of the molecularly defined reference even at a score cut-off value of 2. Also, 412 isolates of 20 different species of non-S. aureus staphylococci were all correctly identified to species level compared to the molecularly defined reference. Moreover, the MALDI-TOF MS-based S. aureus identification approach was clearly faster than more time-consuming methods such as a molecular identification approach.

A histological diagnosis of human intestinal spirochaetosis (HIS) was made in 114 patients during the period 1994–2007. All patients lived in three prefectures in the northern part of Honshu, Japan. Most patients were elderly and male. Twenty-nine patients complained of abdominal pain, bloody stools, diarrhoea or bowel symptoms, but most patients showed no direct symptoms of bowel disease, and occult faecal blood detected at medical check-up was the main reason for colonoscopic examination. There were no homosexual patients and no immunosuppressed patients. HIS was evenly distributed throughout the whole colorectum. PCR analysis of Brachyspira aalborgi and Brachyspira pilosicoli revealed that more patients were infected with B. aalborgi. Follow-up PCR studies confirmed that infestation with B. aalborgi could be repeatedly detected over a 6 year period. This study, involving over 100 patients, identified the characteristic features of HIS in northern Japan. The results suggest that these spirochaetes may be harmless commensals that cause no obvious pathological alterations in infected individuals.

The mercury resistance gene merA has often been found together with antibiotic resistance genes in human commensal Escherichia coli. To study this further, we analysed mercury resistance in collections of strains from various populations with different levels of mercury exposure and various levels of antibiotic resistance. The first population lived in France and had no known mercury exposure. The second lived in French Guyana and included a group of Wayampi Amerindians with a known high exposure to mercury. Carriage rates of mercury resistance were assessed by measuring the MIC and by detecting the merA gene. Mercury-resistant E. coli was found significantly more frequently in the populations that had the highest carriage rates of antibiotic-resistant E. coli and in parallel antibiotic resistance was higher in the population living in an environment with a high exposure to mercury, suggesting a possible co-selection. Exposure to mercury might be a specific driving force for the acquisition and maintenance of mobile antibiotic resistance gene carriage in the absence of antibiotic selective pressure.

This study provides what we believe to be the first report of the presence of EMRSA-15 and its variants isolated from nasal swabs from 13 healthy and diseased individuals in India. The majority of the isolates belonged to staphylococcal cassette chromosome mec (SCCmec) type IV and spa type t852, whilst four isolates were non-typable and heterotypic for the presence of the mecA gene. All non-typable isolates were positive for the orfX gene by PCR and belonged to spa types t005 and t2986. They may have variant SCCmec cassettes indicating genetic changes occurring in the Indian EMRSA-15. All isolates were positive for Panton–Valentine leukocidin and toxic shock syndrome toxin, which is a cause for concern. In addition to soft-tissue infections, the EMRSA-15 isolates from patients were also responsible for meningitis and brain abscesses, which is quite rare.

To investigate the antimicrobial resistance, extended-spectrum β-lactamases (ESBLs) and clones of Klebsiella pneumoniae isolates causing bacteraemia or urinary tract infection (UTI) in Korea, a total of 406 K. pneumoniae isolates from patients with bacteraemia (221 isolates) and UTI (185 isolates) were collected from 10 tertiary-care Korean hospitals from July 2006 to October 2007. In vitro antimicrobial susceptibility testing was performed for all isolates and ESBL production was tested. Multilocus sequence typing (MLST) analyses were performed to characterize genotypes of ESBL-producing K. pneumoniae isolates. PFGE was performed for sequence type 11 (ST11) isolates. Forty-seven UTI isolates (25.4 %) produced ESBLs, while 30 bacteraemia isolates (13.6 %) produced ESBLs (P=0.002). Among 77 ESBL-producing isolates, thirty-two (41.6 %) produced SHV-type ESBLs. bla CTX-M genes such as bla CTX-M-14 and bla CTX-M-15 were detected in 36.4 %. MLST and PFGE analyses showed that ST11 was dominant in ESBL-producing K. pneumoniae isolates causing UTI (57.4 %) and in those causing bacteraemia (70.0 %) and has been prevalent in Korean hospitals. ST11 isolates harbour a combination of different ESBL genes. The ST11 clone of ESBL-producing K. pneumoniae isolates prevails in Korea, but most isolates might acquire ESBL genes independently or several different clones might be distributed in Korea.

Recently vancomycin-resistant and sporadically linezolid-resistant Enterococcus species have been described in adults. We report what we believe to be the first case of a child with prolonged bone marrow aplasia following haematopoietic stem cell transplantation developing a fatal sepsis caused by Enterococcus faecium resistant to glycopeptides and linezolid.

Aeromonas species rarely cause pulmonary infection. We report, for what is believed to be the first time, a case of severe pneumonia in a cancer patient caused by Aeromonas caviae. Detailed microbiological investigation revealed that this isolate carried three β-lactamase-encoding genes (encoding MOX-4, CTX-M-3 and TEM-1) conferring resistance to all β-lactams but imipenem. The β-lactamase with a pI of 9.0 was transferred by conjugation and associated with a 7.3 kb plasmid, as demonstrated by Southern blot hybridization. Analysis of the nucleotide and amino acid sequences showed a new ampC gene that was closely related to those encoding the MOX-1, MOX-2 and MOX-3 β-lactamases. This new plasmid-mediated AmpC β-lactamase from China was named MOX-4. This is believed to be the first report of MOX-4, CTX-M-3 and TEM-1 β-lactamases in a multiresistant A. caviae.

This is the first report, to our knowledge, of two temporally and geographically related nosocomial Asaia lannensis infections in a paediatric setting. Two patients with idiopathic dilated cardiomyopathy awaiting cardiac transplantation developed bacteraemia during their hospital stay. The physical location of both patients, the temporal association of infections, as well as the isolation of two identical pathogens suggested a nosocomial transmission. Commonly used identification methods and instruments failed to identify the isolated pathogens and only 16S rRNA gene sequencing provided definitive identification. These isolates of A. lannensis showed an unfavourable susceptibility pattern including resistance to carbapenems, all β-lactam agents and fluoroquinolones.

Nosocomial infections caused by multidrug-resistant and carbapenem-resistant Pseudomonas putida isolates have been reported occasionally in severely ill or immunocompromised patients. Here we report the microbiological characteristics of what are believed to be the two first carbapenem-resistant VIM metallo-β-lactamase (MBL)-producing P. putida strains in Spain, which were isolated from patients at the University Hospital Complex of Santiago de Compostela. Both patients were immunocompromised with severe underlying diseases and had been hospitalized for more than 15 days. One of them had previously been treated with a broad-spectrum therapy. Antimicrobial susceptibility testing showed that both strains were resistant to piperacillin/tazobactam, ceftazidime, cefepime, imipenem, meropenem, gentamicin, tobramycin, aztreonam, trimethoprim/sulfamethoxazole and ciprofloxacin, but sensitive to amikacin and colistin. For both isolates PCR and sequencing was positive for the bla VIM-2 gene. Fingerprinting analysis revealed these were two different strains. One patient recovered clinically and one died; no direct link could be established between the isolation of P. putida and death. Our data expose the emergence of multidrug-resistant P. putida VIM-2 MBL, probably arising by independent horizontal transfer of resistance genes. So, although P. putida is not frequently isolated, it may survive easily in the hospital setting and occasionally cause difficult-to-treat nosocomial infections in severely ill patients.

A Gram-negative bacterium was isolated from the blood of a patient with diabetes mellitus. However, it could not be identified by conventional microbiological methods, and so was characterized by phenotypic and genotypic analyses. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Neisseria. Based on the phenotypic and genotypic characteristics, we propose that strain SMC-A9199T (=KCTC 22696T=JCM 16127T) should be classified as a novel species, ‘Neisseria skkuensis’ sp. nov. The patient was further treated with amoxicillin–clavulanate and ciprofloxacin for 3 weeks.

A woman was admitted to hospital with abdominal pain. A large kidney stone was recovered and a nephrectomy was performed. Histology revealed the unusual presence of multiple Enterobius vermicularis ova. However, no other parasitic element was recovered on further investigations.