In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA). Among the B. melitensis isolates, the majority (74/82) belonged to MLVA8 genotype 42, clustering in the ‘East Mediterranean’ group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the ‘Americas’ group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal). The majority of B. abortus isolates (51/70) were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.
We investigated the general level of antibiotic resistance with further analysis of extended-spectrum beta-lactamase (ESBL) prevalence, as well as the population structure of E. coli in fecal flora of humans and Franklin’s gulls (Leucophaeus pipixcan) in central parts of Chile. We found a surprisingly high carriage rate of ESBL-producing E. coli among the gulls 112/372 (30.1%) as compared to the human population 6/49 (12.2%.) Several of the E. coli sequence types (STs) identified in birds have previously been reported as Multi Drug Resistant (MDR) human pathogens including the ability to produce ESBLs. This means that not only commensal flora is shared between birds and humans but also STs with pathogenic potential. Given the migratory behavior of Franklin’s gulls, they and other migratory species, may be a part of ESBL dissemination in the environment and over great geographic distances. Apart from keeping the antibiotic use low, breaking the transmission chains between the environment and humans must be a priority to hinder the dissemination of resistance.
The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring blaCTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both blaCTX-M-group-1 and blaOXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, blaOXA-1-like and blaCTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.
Cytochrome P450s are b-heme-containing enzymes that are able to introduce oxygen atoms into a wide variety of organic substrates. They are extremely widespread in nature having diverse functions at both biochemical and physiological level. The genome of C. jejuni 81-176 encodes a single cytochrome P450 (Cj1411c) that has no close homologues. Cj1411c is unusual in its genomic location within a cluster involved in the biosynthesis of outer surface structures. Here we show that E. coli expressed and affinity-purified C. jejuni cytochrome P450 is lipophilic, containing one equivalent Cys-ligated heme. Immunoblotting confirmed the association of cytochrome P450 with membrane fractions. A Cj1411c deletion mutant had significantly reduced ability to infect human cells and was less able to survive following exposure to human serum when compared to the wild type strain. Phenotypically following staining with Alcian blue, we show that a Cj1411c deletion mutant produces significantly less capsular polysaccharide. This study describes the first known membrane-bound bacterial cytochrome P450 and its involvement in Campylobacter virulence.
Although numerous marine bacteria are known to produce antibiotics via hybrid NRPS-PKS gene clusters, none have been previously described in an Alteromonas species. In this study, we describe in detail a novel hybrid NRPS-PKS cluster identified in the plasmid of the Alteromonasmacleodii strain AltDE1 and analyze its relatedness to other similar gene clusters in a sequence-based characterization. This is a mobile cluster, flanked by transposase-like genes, that has even been found inserted into the chromosome of some Alteromonasmacleodii strains. The cluster contains separate genes for NRPS and PKS activity. The sole PKS gene appears to carry a novel acyltransferase domain, quite divergent from those currently characterized. The predicted specificities of the adenylation domains of the NRPS genes suggest that the final compound has a backbone very similar to bleomycin related compounds. However, the lack of genes involved in sugar biosynthesis indicates that the final product is not a glycopeptide. Even in the absence of these genes, the presence of the cluster appears to confer complete or partial resistance to phleomycin, which may be attributed to a bleomycin-resistance-like protein identified within the cluster. This also suggests that the compound still shares significant structural similarity to bleomycin. Moreover, transcriptomic evidence indicates that the NRPS-PKS cluster is expressed. Such sequence-based approaches will be crucial to fully explore and analyze the diversity and potential of secondary metabolite production, especially from increasingly important sources like marine microbes.
The association of PMQR and ESBLs in negative-bacteria isolates has been of great concern. The present study was performed to investigate the prevalence of co-transferability of oqxAB and blaCTX-M genes among the 696 Escherichia coli (E. coli) isolates from food-producing animals in South China, and to characterize these plasmids.
The ESBL-encoding genes (blaCTX-M, blaTEM and blaSHV), and PMQR (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6’)-Ib-cr, qepA, and oqxAB) of these 696 isolates were determined by PCR and sequenced directionally. Conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blotting experiments were performed to investigate the co-transferability and location of oqxAB and blaCTX-M. The EcoRI digestion profiles of the plasmids with oqxAB-blaCTX-M were also analyzed. The clonal relatedness was investigated by PFGE.
Of the 696 isolates, 429 harbored at least one PMQR gene, with oqxAB (328) being the most common type; 191 carried blaCTX-M, with blaCTX-M-14 the most common. We observed a significant higher prevalence of blaCTX-M among the oqxAB-positive isolates (38.7%) than that (17.4%) in the oqxAB-negative isolates. Co-transferability of oqxAB and blaCTX-M was found in 18 of the 127 isolates carrying oqxAB-blaCTX-M. These two genes were located on the same plasmid in all the 18 isolates, with floR being on these plasmids in 13 isolates. The co-dissemination of these genes was mainly mediated by F33:A-: B- and HI2 plasmids with highly similar EcoRI digestion profiles. Diverse PFGE patterns indicated the high prevalence of oqxAB was not caused by clonal dissemination.
blaCTX-M was highly prevalent among the oqxAB-positive isolates. The co-dissemination of oqxAB-blaCTX-M genes in E. coli isolates from food-producing animals is mediated mainly by similar F33:A-: B- and HI2 plasmids. This is the first report of the co-existence of oqxAB, blaCTX-M, and floR on the same plasmids in E. coli.
The largest known outbreak caused by a rare hybrid strain of Shiga toxin-producing E.coli (STEC) and enteroaggregative E. coli (EAEC) (E.coli O104:H4) of serotype O104:H4 occurred in Germany in 2011. Fenugreek sprouts acted as a transmission vehicle and were widely consumed in the outbreak area at the time of the epidemic. In total 3,842 people developed a clinical illness caused by this strain; however the rates of asymptomatic infections remain unclear. We aimed to develop a serological assay for detection of E.coli O104 LPS specific antibodies and to establish the post-outbreak levels of seropositivity among people with documented exposure to contaminated sprouts.
Results and Discussion
Developed serological assays (ELISA with 84% sensitivity, 63% specificity and Western Blot with 100% sensitivity, 82.5% specificity) identified 33% (16/49) level of asymptomatic infection. Relatively small sample size and a significant time- lapse between the onset of symptoms and serum samples collection (appr. 8 weeks) might explain the assay variability. No association was found between clinical or demographic characteristics and assay positivity. Larger studies are needed to understand the complexity of human immune response and factors influencing development of clinical symptoms. Development of intra-outbreak research plans will substantially aid the conduct of more thorough scientific investigation during an outbreak period.
The RcsCDB system of Salmonella enterica serovar Typhimurium is implicated in the control of capsule and flagella synthesis. The hybrid sensor RcsC, the phosphotransferase RcsD and the RcsB regulator, constitute the main components of the RcsCDB system. The proposed Rcs signaling cascade involves the autophosphorylation of RcsC and the transfer of the phosphate group to RcsB, mediated by RcsD. We previously reported that the overexpression of rcsB repress the transcription of rcsD by an autoregulation mechanism. Moreover, we demonstrated that during the rcsD repression, the RcsB-dependent flagellar modulation remained active. These results suggest that the Rcs phosphorelay mechanism occurs even in the absence of RcsD. In this work, we established the existence of two alternative phosphorelay pathways driving activation of this system. We demonstrated that RcsC and RcsD can act as histidine kinase proteins which, after autophosphorylated, are able to independently transfer the phosphate to RcsB. Our results suggest that these pathways could be activated by different environmental signals, leading different levels of RcsB-phosphorylated to produce a differential gene modulation. These findings contribute to a better understanding of the complexity and importance of the Rcs system activation, where more than one phosphate flow pathway increases the possibilities to exert gene regulation for a quick environmental changes response.
To determine factors associated with CTX-M-producing ST131 Escherichia coli which is the worldwide predominant lineage among CTX-M-producing E. coli isolates.
Consecutive inpatients with a clinical sample positive for CTX-M-producing E. coli and considered as cases in a previous 8-month (2008–2009) case-control study performed in ten university hospitals in the Paris area were included in the present sub-population study. Patients with a CTX-M-producing ST131 E. coli clinical isolate were compared with those with a CTX-M-producing non-ST131 E. coli clinical isolate with regard to 66 variables. Variables were first compared using univariate logistic regression, then a multivariate analysis using a backward selection with variables with p-value <0.1 in univariate analysis was carried out.
Fifty-five patients with a CTX-M-producing ST131 E. coli clinical isolate were compared to 97 patients with a CTX-producing non-ST131 E. coli clinical isolate. Multivariate analysis showed that only previous residence in long term care facilities (OR = 4.4; 95% CI = 1.3–14.7) was positively associated with a CTX-M-producing ST131 E. coli isolate. However, it also showed that regular consumption of poultry products (OR = 0.2; 95% CI = 0.1–0.6), having had at least one device in the preceding 6 months (OR = 0.3; 95% CI = 0.1–0.7) and stay in ICU (OR = 0.2; 95% CI = 0.05–0.8) were negatively associated with isolation of CTX-M-producing ST131 E. coli from clinical samples.
This study provides more insight into the epidemiological features of ST131 and non-ST131 E. coli producing CTX-M enzymes. It shows, for the first time, that isolation of CTX-M-producing ST131 E. coli from clinical samples is not linked to consumption of various foods and confirms that residence in long term care facilities is a predictor of these isolates.
A screening for non-target mutations affecting fluoroquinolone susceptibility was conducted in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198. Among a panel of representative isolates (n = 27), covering the epidemic, only three showed distinct mutations in ramR resulting in enhanced expression of genes encoding the AcrAB-TolC efflux system and low increase in ciprofloxacin MIC. No mutations were detected in other regulatory regions of this efflux system. Ciprofloxacin resistance in serovar Kentucky ST198 is thus currently mainly due to multiple target gene mutations.
Salmonella; ciprofloxacin resistance; efflux pump; regulation; ram
A high prevalence of Brucellapinnipedialis serology and bacteriology positive animals has been found in the Northeast Atlantic stock of hooded seal (Cystophoracristata); however no associated gross pathological changes have been identified. Marine mammal brucellae have previously displayed different infection patterns in human and murine macrophages. To investigate if marine mammal Brucella spp. are able to invade and multiply in cells originating from a presumed host species, we infected alveolar macrophages from hooded seal with a B. pinnipedialis hooded seal isolate. Hooded seal alveolar macrophages were also challenged with B. pinnipedialis reference strain (NCTC 12890) from harbor seal (Phocavitulina), B. ceti reference strain (NCTC 12891) from harbor porpoise (Phocoenaphocoena) and a B. ceti Atlantic white-sided dolphin (Lagenorhynchusacutus) isolate (M83/07/1), to evaluate possible species-specific differences. Brucella suis 1330 was included as a positive control. Alveolar macrophages were obtained by post mortem bronchoalveolar lavage of euthanized hooded seals. Phenotyping of cells in the lavage fluid was executed by flow cytometry using the surface markers CD14 and CD18. Cultured lavage cells were identified as alveolar macrophages based on morphology, expression of surface markers and phagocytic ability. Alveolar macrophages were challenged with Brucella spp. in a gentamicin protection assay. Following infection, cell lysates from different time points were plated and evaluated quantitatively for colony forming units. Intracellular presence of B. pinnipedialis hooded seal isolate was verified by immunocytochemistry. Our results show that the marine mammal brucellae were able to enter hooded seal alveolar macrophages; however, they did not multiply intracellularly and were eliminated within 48 hours, to the contrary of B. suis that showed the classical pattern of a pathogenic strain. In conclusion, none of the four marine mammal strains tested were able to establish a persistent infection in primary alveolar macrophages from hooded seal.
This paper covers eight Salmonella serogroups, that are defined by O antigens with related structures and gene clusters. They include the serovars that are now most frequently isolated. Serogroups A, B1, B2, C2-C3, D1, D2, D3 and E have O antigens that are distinguished by having galactose as first sugar, and not N-acetyl glucosamine or N-acetyl galactosamine as in the other 38 serogroups, and indeed in most Enterobacteriaceae. The gene clusters for these galactose-initiated appear to have entered S. enterica since its divergence from E. coli, but sequence comparisons show that much of the diversification occurred long before this. We conclude that the gene clusters must have entered S. enterica in a series of parallel events. The individual gene clusters are discussed, followed by analysis of the divergence for those genes shared by two or more gene clusters, and a putative phylogenic tree for the gene clusters is presented. This set of O antigens provides a rare case where it is possible to examine in detail the relationships of a significant number of O antigens. In contrast the more common pattern of O-antigen diversity within a species is for there to be only a few cases of strains having related gene clusters, suggesting that diversity arose through gain of individual O-antigen gene clusters by lateral gene transfer, and under these circumstances the evolution of the diversity is not accessible. This paper on the galactose-initiated set of gene clusters gives new insights into the origins of O-antigen diversity generally.
Administration of antibiotics to food animals may select for drug-resistant pathogens of clinical significance, such as methicillin-resistant Staphylococcus aureus (MRSA). In the United States, studies have examined prevalence of MRSA carriage among individuals exposed to livestock, but prevalence of multidrug-resistant S. aureus (MDRSA) carriage and the association with livestock raised with versus without antibiotic selective pressure remains unclear. We aimed to examine prevalence, antibiotic susceptibility, and molecular characteristics of S. aureus among industrial livestock operation (ILO) and antibiotic-free livestock operation (AFLO) workers and household members in North Carolina.
Participants in this cross-sectional study were interviewed and provided a nasal swab for S. aureus analysis. Resulting S. aureus isolates were assessed for antibiotic susceptibility, multi-locus sequence type, and absence of the scn gene (a marker of livestock association).
Among 99 ILO and 105 AFLO participants, S. aureus nasal carriage prevalence was 41% and 40%, respectively. Among ILO and AFLO S. aureus carriers, MRSA was detected in 7% (3/41) and 7% (3/42), respectively. Thirty seven percent of 41 ILO versus 19% of 42 AFLO S. aureus-positive participants carried MDRSA. S. aureus clonal complex (CC) 398 was observed only among workers and predominated among ILO (13/34) compared with AFLO (1/35) S. aureus-positive workers. Only ILO workers carried scn-negative MRSA CC398 (2/34) and scn-negative MDRSA CC398 (6/34), and all of these isolates were tetracycline resistant.
Despite similar S. aureus and MRSA prevalence among ILO and AFLO-exposed individuals, livestock-associated MRSA and MDRSA (tetracycline-resistant, CC398, scn-negative) were only present among ILO-exposed individuals. These findings support growing concern about antibiotics use and confinement in livestock production, raising questions about the potential for occupational exposure to an opportunistic and drug-resistant pathogen, which in other settings including hospitals and the community is of broad public health importance.
Variable number of tandem repeats (VNTRs) that are widely distributed in the genome of Yersinia pestis proved to be useful markers for the genotyping and source-tracing of this notorious pathogen. In this study, we probed into the features of VNTRs in the Y. pestis genome and developed a simple hierarchical genotyping system based on optimized VNTR loci.
Capillary electrophoresis was used in this study for multi-locus VNTR analysis (MLVA) in 956 Y. pestis strains. The general features and genetic diversities of 88 VNTR loci in Y. pestis were analyzed with BioNumerics, and a “14+12” loci-based hierarchical genotyping system, which is compatible with single nucleotide polymorphism-based phylogenic analysis, was established.
Appropriate selection of target loci reduces the impact of homoplasies caused by the rapid mutation rates of VNTR loci. The optimized “14+12” loci are highly discriminative in genotyping and source-tracing Y. pestis for molecular epidemiological or microbial forensic investigations with less time and lower cost. An MLVA genotyping datasets of representative strains will improve future research on the source-tracing and microevolution of Y. pestis.
Infections caused by Extended spectrum β-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used β-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements. We have characterized the plasmid content of ESBL-producing E. coli from human urinary tract infections. Ten diverse isolates were selected; they had unrelated pulsed-field gel electrophoresis (PFGE) types (<90% similarity), were from geographically dispersed locations and had diverging antibiotic resistance profiles. Three isolates belonged to the globally disseminated sequence type ST131. ESBL-genes of the CTX-M-1 and CTX-M-9 phylogroups were identified in all ten isolates. The plasmid content (plasmidome) of each strain was analyzed using a combination of molecular methods and high-throughput sequencing. Hidden Markov Model-based analysis of unassembled sequencing reads was used to analyze the genetic diversity of the plasmid samples and to detect resistance genes. Each isolate contained between two and eight distinct plasmids, and at least 22 large plasmids were identified overall. The plasmids were variants of pUTI89, pKF3-70, pEK499, pKF3-140, pKF3-70, p1ESCUM, pEK204, pHK17a, p083CORR, R64, pLF82, pSFO157, and R721. In addition, small cryptic high copy-number plasmids were frequent, containing one to seven open reading frames per plasmid. Three clustered groups of such small cryptic plasmids could be distinguished based on sequence similarity. Extrachromosomal prophages were found in three isolates. Two of them resembled the E. coli P1 phage and one was previously unknown. The present study confirms plasmid multiplicity in multi-resistant E. coli. We conclude that high-throughput sequencing successfully provides information on the extrachromosomal gene content and can be used to generate a genetic fingerprint of possible use in epidemiology. This could be a valuable tool for tracing plasmids in outbreaks.
The prevalence of Salmonella enterica serovar Enteritidis is gradually decreasing in poultry flocks in the EU, which may result in the demand for a vaccine that allows for the differentiation of vaccinated flocks from those infected by wild-type S. Enteritidis. In this study, we therefore constructed a (Salmonella Pathogenicity Island 1) SPI1-lon mutant with or without fliC encoding for S. Enteritidis flagellin. The combination of SPI1-lon mutations resulted in attenuated but immunogenic mutant suitable for oral vaccination of poultry. In addition, the vaccination of chickens with the SPI1-lon-fliC mutant enabled the serological differentiation of vaccinated and infected chickens. The absence of fliC therefore did not affect the immunogenicity of the vaccine strain and allowed for serological differentiation of the vaccinated chickens. The SPI1-lon-fliC mutant is therefore a suitable marker vaccine strain for oral vaccination of poultry.
The increased prevalence of multidrug-resistant (MDR) bacteria in combination with the relatively limited development of new antibiotics presents a serious threat to public health. In chicken, especially Extended-Spectrum ß-Lactamase (ESBL) carrying Enterobacteriaceae are often asymptomatically present but can infect humans. Due to their broad range antimicrobial activity cathelicidins and other host defence peptides, are considered to be an attractive alternative to conventional antibiotics. In this study, the antimicrobial activity of three chicken cathelicidins against a broad array of multidrug resistant bacteria was determined. All three peptides showed high antibacterial activity independent of the presence of MDR characteristics. Induction experiments using S. aureus and K. pneumoniae showed that although an increase in resistance was initially observed, susceptibility towards chicken cathelicidins remained high and no major resistance was developed. The combined results underline the potential of chicken cathelicidins as a new alternative to antibiotics.
The NDM-1 carbapenemase has been identified in 2008 in Enterobacteriaceae. Since then, several reports have emphasized its rapid dissemination throughout the world. The spread of NDM carbapenemases involve several blaNDM gene variants associated with various plasmids among several Gram negative species.
A multidrug-resistant E. coli isolate recovered from urine of a patient who had travelled to Burma has been characterized genetically and biochemically.
E. coli COU was resistant to all antibiotics tested except amikacin, tigecycline, fosfomycin, and chloramphenicol. Analysis of the antibiotic resistance traits identified a metallo-ß-lactamase, a novel NDM variant, NDM-7. It differs from NDM-4 by a single amino acid substitution sharing an identical extended spectrum profile towards carbapenems. The blaNDM-7 gene was located on an untypeable conjugative plasmid and associated with a close genetic background similar to those described among the blaNDM-1 genes. The isolate also harbours blaCTXM-15 and blaOXA-1 genes and belonged to ST167.
This study highlights that spread of NDM producers correspond to spread of multiple blaNDM genes and clones and therefore will be difficult to control.
Fluoroquinolones are very important drugs in the clinical antibacterial arsenal; their success is principally due to their mode of action: the stabilisation of a gyrase-DNA intermediate (the cleavage complex), which triggers a chain of events leading to cell death. Microcin B17 (MccB17) is a modified peptide bacterial toxin that acts by a similar mode of action, but is unfortunately unsuitable as a therapeutic drug. However, its structure and mechanism could inspire the design of new antibacterial compounds that are needed to circumvent the rise in bacterial resistance to current antibiotics. Here we describe the investigation of the structural features responsible for MccB17 activity and the identification of fragments of the toxin that retain the ability to stabilise the cleavage complex.
Salmonella genomic island 1 (SGI1) is a 43-kb integrative mobilizable element that harbors a great diversity of multidrug resistance gene clusters described in numerous Salmonella enterica serovars and also in Proteus mirabilis. The majority of SGI1 variants contain an In104-derivative complex class 1 integron inserted between resolvase gene res and open reading frame (ORF) S044 in SGI1. Recently, the international spread of ciprofloxacin-resistant S. enterica serovar Kentucky sequence type 198 (ST198) containing SGI1-K variants has been reported. A retrospective study was undertaken to characterize ST198 S. Kentucky strains isolated before the spread of the epidemic ST198-SGI1-K population in Africa and the Middle East. Here, we characterized 12 ST198 S. Kentucky strains isolated between 1969 and 1999, mainly from humans returning from Southeast Asia (n = 10 strains) or Israel (n = 1 strain) or from meat in Egypt (n = 1 strain). All these ST198 S. Kentucky strains did not belong to the XbaI pulsotype X1 associated with the African epidemic clone but to pulsotype X2. SGI1-J subgroup variants containing different complex integrons with a partial transposition module and inserted within ORF S023 of SGI1 were detected in six strains. The SGI1-J4 variant containing a partially deleted class 1 integron and thus showing a narrow resistance phenotype to sulfonamides was identified in two epidemiologically unrelated strains from Indonesia. The four remaining strains harbored a novel SGI1-J variant, named SGI1-J6, which contained aadA2, floR2, tetR(G)-tetA(G), and sul1 resistance genes within its complex integron. Moreover, in all these S. Kentucky isolates, a novel insertion sequence related to the IS630 family and named ISSen5 was found inserted upstream of the SGI1 complex integron in ORF S023. Thus, two subpopulations of S. Kentucky ST198 independently and exclusively acquired the SGI1 during the 1980s and 1990s. Unlike the ST198-X1 African epidemic subpopulation, the ST198-X2 subpopulation mainly from Asia harbors variants of the SGI1-J subgroup that are encountered mainly in the Far East, as previously described for S. enterica serovars Emek and Virchow.
Recently, novel Brucella strains with phenotypic characteristics that were atypical for strains belonging to the genus Brucella have been reported. Phenotypically many of these strains were initially misidentified as Ochrobactrum spp. Two novel species have been described so far for these strains, i.e., B. microti and B. inopinata, and other strains genetically related to B. inopinata may constitute other novel species as well. In this study, we analyzed the lipopolysaccharides (LPS) (smooth LPS [S-LPS] and rough LPS [R-LPS]) of these atypical strains using different methods and a panel of monoclonal antibodies (MAbs) directed against several epitopes of the Brucella O-polysaccharide (O-PS) and R-LPS. Among the most striking results, Brucella sp. strain BO2, isolated from a patient with chronic destructive pneumonia, showed a completely distinct S-LPS profile in silver stain gels that looked more similar to that of enterobacterial S-LPS. This strain also failed to react with MAbs against Brucella O-PS epitopes and showed weak reactivity with anti-R-LPS MAbs. B. inopinata reference strain BO1 displayed an M-dominant S-LPS type with some heterogeneity relative to the classical M-dominant Brucella S-LPS type. Australian wild rodent strains belonging also to the B. inopinata group showed a classical A-dominant S-LPS but lacked the O-PS common (C) epitopes, as previously reported for B. suis biovar 2 strains. Interestingly, some strains also failed to react with anti-R-LPS MAbs, such as the B. microti reference strain and B. inopinata BO1, suggesting modifications in the core-lipid A moieties of these strains. These results have several implications for serological typing and serological diagnosis and underline the need for novel tools for detection and correct identification of such novel emerging Brucella spp.
Four methicillin-resistant coagulase-negative staphylococci (MRCoNS), one Staphylococcus haemolyticus and three Staphylococcus cohnii, from infections of humans collected via the Ministry of Health National Antimicrobial Resistance Surveillance Net (Mohnarin) program in China were identified as linezolid-resistant. These four isolates were negative for the 23S rRNA mutations, but positive for the gene cfr. Mutations in the gene for the ribosomal protein L3, which resulted in the amino acid exchanges Gly152Asp and Tyr158Phe, were identified in S. haemolyticus 09D279 and S. cohnii NDM113, respectively. In each isolate, the cfr gene was located on a plasmid of ca. 35.4 kb, as shown by S1 nuclease pulsed-field gel electrophoresis and Southern blotting experiments. This plasmid was indistinguishable from the previously described plasmid pSS-02 by its size, restriction pattern, and a sequenced 14-kb cfr-carrying segment. Plasmid pSS-02 was originally identified in staphylococci isolated from pigs. This is the first time that a cfr-carrying plasmid has been detected in MRCoNS obtained from intensive care patients in China. Based on the similarities to the cfr-carrying plasmid pSS-02 from porcine coagulase-negative staphylococci, a transmission of this cfr-carrying plasmid between staphylococci from pigs and humans appears to be likely.
The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.
Clostridium difficile are Gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15–35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking.
Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production.
This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.
The brucellae are Gram-negative bacteria that cause an important zoonosis. Studies with the main Brucella species have shown that the O-antigens of the Brucella smooth lipopolysaccharide are α-(1→2) and α-(1→3)-linked N-formyl-perosamine polysaccharides that carry M, A and C (A = M, A>M and AA) and M specificities. However, the biovar 2 O-antigen bound monoclonal antibodies to the Brucella A epitope, and to the C/Y epitope shared by brucellae and Yersinia enterocolitica O:9, a bacterium that carries an N-formyl-perosamine O-antigen in exclusively α-(1→2)-linkages. By 13C NMR spectroscopy, B. suis biovar 1 but not B. suis biovar 2 or Y. enterocolitica O:9 polysaccharide showed the signal characteristic of α-(1→3)-linked N-formyl-perosamine, indicating that biovar 2 may altogether lack this linkage. Taken together, the NMR spectroscopy and monoclonal antibody analyses strongly suggest a role for α-(1→3)-linked N-formyl-perosamine in the C (A = M) and C (M>A) epitopes. Moreover, they indicate that B. suis biovar 2 O-antigen lacks some lipopolysaccharide epitopes previously thought to be present in all smooth brucellae, thus representing a new brucella serovar that is M-negative, C-negative. Serologically and structurally this new serovar is more similar to Y. enterocolitica O:9 than to other brucellae.