- Volume 57, Issue 8, 2008

The amount of lipopolysaccharide (LPS) O antigen (OAg) and its chain length distribution are important factors that protect bacteria from serum complement. Salmonella enterica serovar Typhi produces LPS with long chain length distribution (L-OAg) controlled by the wzz gene, whereas serovar Typhimurium produces LPS with two OAg chain lengths: an L-OAg controlled by WzzST and a very long (VL) OAg determined by WzzfepE. This study shows that serovar Enteritidis also has a bimodal OAg distribution with two preferred OAg chain lengths similar to serovar Typhimurium. It was reported previously that OAg production by S. Typhi increases at the late exponential and stationary phases of growth. The results of this study demonstrate that increased amounts of L-OAg produced by S. Typhi grown to stationary phase confer higher levels of bacterial resistance to human serum. Production of OAg by serovars Typhimurium and Enteritidis was also under growth-phase-dependent regulation; however, while the total amount of OAg increased during growth, the VL-OAg distribution remained constant. The VL-OAg distribution was primarily responsible for complement resistance, protecting the non-typhoidal serovars from the lytic action of serum irrespective of the growth phase. As a result, the non-typhoidal species were significantly more resistant than S. Typhi to human serum. When S. Typhi was transformed with a multicopy plasmid containing the S. Typhimurium wzz fepE gene, resistance to serum increased to levels comparable to the non-typhoidal serovars. In contrast to the relevant role for high-molecular-mass OAg molecules, the presence of Vi antigen did not contribute to serum resistance of clinical isolates of serovar Typhi.

In this study we evaluated the performance of the oxacillin agar screen test, and agar dilution tests using cefoxitin and oxacillin antimicrobials, to detect meticillin resistance in Staphylococcus aureus isolates. The presence of the mecA gene, detected by PCR, was used as the standard to which agar screen and agar dilution tests were compared. The best performance was obtained using the agar dilution test (99.4 % accuracy) with breakpoints of 4 μg ml−1 for oxacillin and 8 μg ml−1 for cefoxitin, and using the oxacillin agar screen test. Also, a strong correlation between MIC values of cefoxitin and oxacillin permits the use of either drug for detection of meticillin resistance.

Staphylococci are the main causative agents of nosocomial diseases. Over the last few years, the increase in the number of meticillin-resistant (MR) staphylococci has become a major clinical problem. Accuracy and promptness in the detection of meticillin resistance are of key importance in ensuring the correct antibiotic treatment in infected patients and control of MR staphylococci in the hospital environment. This study evaluated the accuracy of a cefoxitin disc diffusion (DD) test for the detection of meticillin resistance in staphylococci. A total of 144 clinical isolates [97 Staphylococcus aureus and 47 coagulase-negative staphylococci (CoNS)] were tested using a mecA gene PCR, a DD test (oxacillin, 1 μg disc; cefoxitin, 30 μg disc), determination of oxacillin MIC by agar dilution (AD), and an oxacillin screen agar test at oxacillin concentrations of 4 and 6 μg ml−1. Of the 97 S. aureus and 47 CoNS isolates, 73 (75.26 %) and 30 (63.83 %), respectively, were mecA-positive. The sensitivity and specificity of the cefoxitin DD test were 94.44 and 95.83 %, respectively, for S. aureus and 80 and 100 %, respectively, for CoNS. The oxacillin DD method was 100 % sensitive and 58.33 % specific for S. aureus, and 86.67 % sensitive and 70.59 % specific for CoNS. The AD test was highly sensitive (98.63 %) and specific (98.53 %) for S. aureus and CoNS (83.33 % sensitive and 94.12 % specific). The cefoxitin DD test for meticillin-resistance detection was more specific but less sensitive than the oxacillin DD test. Use of DD tests for both cefoxitin and oxacillin can help in more accurate prediction of meticillin resistance. Centres that are not equipped to carry out PCR can use AD methods for confirmation of meticillin resistance, especially in oxacillin-resistant and cefoxitin-sensitive cases.

A recent outbreak of lymphogranuloma venereum (LGV) within the men who have sex with men (MSM) community and their requirement for extended therapy has highlighted the need for laboratory tests that differentiate LGV- from non-LGV-associated serovars of Chlamydia trachomatis. Two previously described methods were evaluated against 495 clinical specimens referred to the Sexually Transmitted Bacteria Reference Laboratory (London, UK): (i) PCR amplification of a 1.1 kb region of the ompI gene followed by restriction enzyme digestion (ompI RFLP-PCR); and (ii) real-time PCR targeting a 36 bp deletion present within the polymorphic membrane protein H gene of LGV-associated serovars (pmpH real-time PCR). For specimens that could be categorized using both methods, a 94.7 % (390/412) concordance was achieved. Eighty-three specimens were found to be untypeable by ompI RFLP-PCR due to a failure to amplify the 1.1 kb fragment. Of these 83 untypeable specimens, 19 were determined to be an LGV-associated serovar by pmpH real-time PCR. Despite the high level of concordance, there were differences found in the technical complexity of the two methods. The pmpH real-time PCR exhibited greater sensitivity, a more rapid turnaround time and a lower technical requirement. Whilst the ompI RFLP-PCR was not as robust as a laboratory diagnostic method, it did enable serovar-level identification. LGV infection remains an important threat to the health of high-risk MSM in Europe. In conclusion, the two methods for the detection of LGV from clinical samples were found not only to have a high concordance (94.7 %) but also to be complementary, and could be used in an integrated way to aid LGV detection.

Limited β-lactams show antipseudomonal activity. The rapid spread of IMP-type metallo-β-lactamases (MBLs), which have a broad spectrum of substrates and a poor susceptibility to clinically available inhibitors, further restricts β-lactam use. In the present study, we evaluated the potency of IMP-10 MBL in hydrolysing antipseudomonal β-lactams currently available in the clinic. Crude IMP-10 MBL was prepared from two clinical isolates of Pseudomonas aeruginosa harbouring the bla IMP-10 gene. The sensitivity of β-lactams to hydrolysis by IMP-10 MBL was determined by comparing the MICs of 14 antipseudomonal β-lactams against a susceptible strain of P. aeruginosa in the presence and absence of IMP-10 MBL. Carbapenems (imipenem, meropenem and panipenem) and extended-spectrum cephems (ceftazidime, cefoperazone, cefsulodin and cefepime) were sensitive to the hydrolysing activity of IMP-10 MBL. By comparison, the fourth-generation cephem (cefpirome), the extended-spectrum penicillins (carbenicillin, ticarcillin, piperacillin and mezlocillin) and monobactams (aztreonam and carumonam) were relatively resistant to IMP-10 MBL. The sensitivity profile of antipseudomonal β-lactams to IMP-10 MBL generated in the present study provides a valuable reference for antibiotic selection by medical professionals.

Human granulocytic anaplasmosis (HGA) and Lyme borreliosis (LB) are tick-borne infectious diseases caused by Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato species, respectively. In this study, p44/msp2 paralogues specific to A. phagocytophilum and 5S–23S rRNA gene-intergenic spacers specific to B. burgdorferi sensu lato species were detected by PCR in ticks collected in two regions, Tver (Kalinin) and Konakovo, of the Tver (Kalinin) Province located 150 km north-west of Moscow. The PCR amplicons obtained were further characterized by sequencing and RFLP analysis. In the total of 199 ticks collected, 8.8 % (7/80) and 33.8 % (27/80) of Ixodes ricinus, and 2.5 % (3/119) and 45.4 % (54/119) of Ixodes persulcatus, were found to be infected with A. phagocytophilum and B. burgdorferi sensu lato spp., respectively. Of those 199 ticks, 5 (2.5 %) were coinfected with A. phagocytophilum and Borrelia afzelii. Phylogenetic analysis revealed unique p44/msp2 paralogous genes in A. phagocytophilum-infected Russian ticks. The sequence similarities with those of A. phagocytophilum in the United States, UK and Japan ranged from 42 % to 80.4 %, and there were no sequences showing more than 90 % similarity with those sequences from the other countries. The results showed that the p44/msp2 sequence similarity groups may provide an index of adaptation of A. phagocytophilum strains to specific vector ticks or reservoir hosts in different countries and areas. These findings suggest that there is a public health threat from HGA and LB in Tver Province surrounding Moscow.

The penicillin-resistant Spain9V-3 clone of Streptococcus pneumoniae is widespread and presents different serotype variants originating from recombination of the capsular genes. In this work, the genetic relatedness of 29 invasive pneumococci isolated from the central region of Argentina (Cordoba, Buenos Aires, Santa Fe and La Pampa provinces) was assessed by multilocus sequence typing (MLST). All of the penicillin-non-susceptible isolates studied (21/29) belonged to a serotype 14 variant of the Spain9V-3 clone. This clone was predominant, suggesting that it was responsible for the penicillin resistance spread in this region. Interestingly, this serotype 14 variant (named Cordoba S14V) could be differentiated from the European one by its pbp1a gene, suggesting a different recombinational replacement of the capsular genes. The putative recombination sites were analysed, resulting in the proximal crossover point being clearly localized in the spr0309 gene, with the distal site restricted to the recU gene, confirming a different recombination event. Analysis of the dexB, cpsB, aliA and pbp1a genes from these strains showed a high similarity with the corresponding genes of the Spain14-5 clone, suggesting that the capsular genes were provided by this international clone. Analysis of the genetic polymorphisms of the pbp1a (nt 1473–1922) and spr0309 (nt 1–790) genes is proposed as an epidemiological tool to help recognize the Cordoba S14V of the Spain9V-3 clone. On the other hand, BOX-repeat-based PCR and MLST analyses of serotype 14 strains revealed a divergent epidemiology of the Cordoba S14V, suggesting a non-recent dissemination in the paediatric population. It is suggested that this molecular epidemiology work will be a reference for monitoring the evolution of S14Vs of Spain9V-3, the emergence of new clones and the impact of pneumococcal vaccination programmes in Argentina.

We evaluated the relationship between the intestinal microbiota composition and clinical outcome in a group of 15 high-risk patients admitted for acute infection and/or surgical/accidental trauma who were treated with systemic antibiotics according to standard intensive care unit (ICU) protocols. There was a high mortality rate amongst these patients, each of whom had a considerable organ failure score at admission, respiratory assistance during the most of their ICU stay and a long length of stay. All of these individuals received sedation and enteral nutrition, and the majority also received insulin, vasoactive drugs and some stress-ulcer prophylaxis agents. The intestinal microbiota composition was assessed using denaturing gradient gel electrophoresis (DGGE), a molecular biology tool used to characterize bacterial ecosystems. As all of the patient subjects were in good health prior to their acute illness and admission to the ICU, the first faecal samples obtained from this group showed a DGGE banding pattern that was similar to that of healthy subjects. After 1 week of critical illness, coupled with intensive care treatment, including antibiotics, a very definite alteration in the overall microbiota composition was evident, as revealed by a reduction in the number of DGGE bands. Further pronounced changes to the DGGE banding profiles could be observed in patients remaining in the ICU for 2 weeks. Moreover, a dominant band, identified by sequencing as highly related to Enterococcus, was detected in the DGGE profile of some of our patient subjects. We also performed real-time PCR and obtained results that were in agreement with our qualitative evaluations using DGGE. The degree of organ failure and ICU mortality was significantly higher in patients for whom a high reduction in microbiota biodiversity was coupled with a massive presence of enterococci. A statistically significant link between these two ecological traits and the use of clindamycin was also found.

This study compared the rate of recovery of meticillin-resistant Staphylococcus aureus (MRSA) between the periods 2001–2003 and 2004–2006 in acute and chronic maxillary sinusitis. Cultures were obtained from 458 patients, 244 with acute and 214 with chronic maxillary sinusitis; 215 isolates were recovered in the 2 years between 2001 and 2003 (118 from acute and 97 from chronic sinusitis), and 243 in the 2 years between 2004 and 2006 (126 from acute and 117 from chronic sinusitis). S. aureus was isolated from ten (8 %) of the patients with acute sinusitis between 2001 and 2003, three (30 %) of which were MRSA, and from 13 (10 %) of the patients with acute sinusitis between 2004 and 2006, nine (69 %) of which were MRSA (P <0.01). S. aureus was found in 15 (15 %) of the patients with chronic sinusitis between 2001 and 2003, four (27 %) of which were MRSA, and from 23 (20 %) of the patients with chronic sinusitis between 2004 and 2006, 14 (61 %) of which were MRSA (P <0.05). Antimicrobial therapy was administered over the last 3 months to 122 (57 %) of the patients with chronic sinusitis. MRSA was isolated more often from these individuals (28/122; 23 %) than from those not treated previously (10/92 or 11 %) (P <0.05). These data illustrate that a significant increase occurred in the rate of recovery of MRSA in patients with acute and chronic maxillary sinusitis over the periods studied.

The biofilm-forming capacity of 972 clinical isolates of Staphylococcus aureus was tested using a high-throughput polystyrene 96-peg plate format. Isolates of S. aureus were collected from patients in hospitals throughout Scotland from 2004 to 2006; 763 of these were meticillin-resistant S. aureus (MRSA) and 209 were meticillin-sensitive S. aureus (MSSA). The biomass of each biofilm was quantified using a crystal violet staining technique. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The majority of MRSA (53.8 %) and MSSA (43.5 %) isolates formed moderately attached biofilms. Fully established biofilms were formed by 20.5 % of MRSA isolates and 28.0 % of MSSA isolates, whilst 25.7 % of MRSA isolates and 28.5 % of MSSA isolates formed negligible biofilms. <-- INSERT SHAPE --><-- INSERT SHAPE -->There was no significant correlation between susceptibility to meticillin and biofilm formation (P=0.77). MRSA isolates were divided into clonal types (EMRSA-15, EMRSA-16 and sporadic isolates) based on PFGE genotyping results. EMRSA-15 isolates formed significantly more moderately and fully established biofilms than EMRSA-16 isolates (P<0.001). S. aureus strains isolated from the skin of patients had a significantly greater capacity to form biofilms than isolates from other body sites, including the blood. Microscopic examination of biofilms by scanning electron microscopy (SEM) revealed that poorly adherent biofilm formers failed to colonize the entire surface of the peg, whilst moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group that formed fully established biofilms confirmed that this isolate developed a dense biofilm with a textured, multi-layered, three-dimensional structure.

We have evaluated two simple, rapid and low-cost colorimetric methods for the detection of multidrug-resistant Mycobacterium tuberculosis. A total of 151 M. tuberculosis strains were tested for resistance to rifampicin (RMP) and isoniazid by resazurin microplate assay (REMA) and nitrate reductase assay (NRA) in comparison with the conventional proportion method (PM) on Löwenstein-Jensen medium. A complete agreement was found between NRA and PM, while one false RMP-susceptible result was found by REMA. REMA and NRA tests are rapid and inexpensive, and could be good alternatives to the conventional PM in low-resource countries.

Anaemia is frequent in patients with human immunodeficiency virus infection, and antiretroviral drugs have been implicated. Whilst therapy-induced anaemia is more readily attributed to zidovudine, lamivudine-associated, potentially life-threatening, pure red cell aplasia (PRCA) has been less recognized in the past and is only infrequently documented in the literature. We report on a patient suffering from what turned out to be lamivudine-induced PRCA who required 15 units of blood within 3 weeks before recovering swiftly following lamivudine (3TC) treatment withdrawal. As reviewed here, the nature of this condition is not well described in general, the onset appears to be variable and occurs at any CD4+ count, but rapid improvement after cessation of drug administration appears to be a consistent feature.

Daptomycin is a novel bactericidal agent active against Gram-positive pathogens including meticillin-resistant Staphylococcus aureus (MRSA). Our case is unique in the description of an MRSA USA 300 isolate that developed decreased susceptibility to daptomycin during daptomycin and vancomycin therapy. Directed sequencing detected a previously reported mutation in mprF, resulting in a T345A substitution, associated with non-susceptibility to daptomycin.