Epidemiology and molecular typing of multidrug-resistant bacteria in tertiary hospitals and nursing homes in Flanders, Belgium.

This study aimed to map MDRO carriage and potential transmission within and between three Flemish tertiary care hospitals and their neighbouring nursing homes. A cross-sectional MDRO prevalence survey was organized between October 2017 and February 2019. Perianal swabs were cultured for detection of MDRO. Determination of clonal relatedness based on wgMLST allelic profiles was performed. The prevalence of MDRO in Belgian hospitals and NHs is on the rise, compared to previous studies, and transmission in and between institutions is observed. These results re-emphasize the need for a healthcare network-wide infection prevention strategy in which WGS of MDRO strains can be supportive.

Epidemiology and molecular typing of multidrug-resistant bacteria in day care centres in Flanders, Belgium.

The global prevalence and spread of multidrug-resistant organisms (MDROs) represent an emerging public health threat. Day care centre (DCC) attendance is a risk factor for MDRO carriage in children and their environment. This study aimed to map the epidemiology of carriage and potential transmission of these organisms within 18 Flemish DDCs (Belgium). An MDRO prevalence survey was organised between November 2018 and February 2019 among children attending the centres. Selective chromogenic culture media were used for the detection of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), and vancomycin-resistant Enterococci (VRE) in faecal swabs obtained from diapers or jars (n = 448). All isolated MDROs were subjected to resistance gene sequencing. A total of 71 of 448 samples (15.8%) yielded isolates of ESBL-E with a predominance of Escherichia coli (92.2% of ESBL-E) and ESBL resistance gene blaCTX-M-15 (50.7% of ESBL coding genes in E. coli). ESBL-E prevalence varied between DCCs, ranging from 0 to 50%. Transmission, based on the clonal relatedness of ESBL-E strains, was observed. CPE was identified in only one child carrying an E. coli with an OXA-244 gene. VRE was absent from all samples. The observed prevalence of ESBL-E in Flemish DCCs is high compared with previous studies, and our findings re-emphasise the need for rigorous hygiene measures within such centres to control the further spread of MDROs in the community.

Genetic characterization of ESBL-producing and ciprofloxacin-resistant Escherichia coli from Belgian broilers and pigs.

Background: The increasing number of infections caused by Escherichia coli resistant to clinically important antibiotics is a global concern for human and animal health. High overall levels of extended-spectrum beta-lactamase (ESBL)-producing and ciprofloxacin-resistant (ciproR) Escherichia coli in livestock are reported in Belgium. This cross-sectional study aimed to genotypically characterize and trace ESBL-and ciproR-E. coli of Belgian food-producing animals. Methods: A total of 798 fecal samples were collected in a stratified-random sampling design from Belgian broilers and sows. Consequently, 77 ESBL-E. coli and 84 ciproR-E. coli were sequenced using Illumina MiSeq. Minimum inhibitory concentration (MIC) for fluoroquinolones and cephalosporins were determined. Molecular in silico typing, resistance and virulence gene determination, and plasmid identification was performed. Scaffolds harboring ESBL or plasmid-mediated quinolone resistance (PMQR) genes were analyzed to detect mobile genetic elements (MGEs) and plasmid origins. Core genome allelic distances were used to determine genetic relationships among isolates. Results: A variety of E. coli sequence types (ST) (n = 63), resistance genes and virulence profiles was detected. ST10 was the most frequently encountered ST (8.1%, n = 13). The pandemic multidrug-resistant clone ST131 was not detected. Most farms harbored more than one ESBL type, with bla CTX-M-1 (41.6% of ESBL-E. coli) being the most prevalent and bla CTX M-15 (n = 3) being the least prevalent. PMQR genes (15.5%, n = 13) played a limited role in the occurrence of ciproR-E. coli. More importantly, sequential acquisition of mutations in quinolone resistance-determining regions (QRDR) of gyrA and parC led to increasing MICs for fluoroquinolones. GyrA S83L, D87N and ParC S80I mutations were strongly associated with high-level fluoroquinolone resistance. Genetically related isolates identified within the farms or among different farms highlight transmission of resistant E. coli or the presence of a common reservoir. IncI1-I(alpha) replicon type plasmids carried different ESBL genes (bla CTX-M-1, bla CTX-M-32 and bla TEM-52C). In addition, the detection of plasmid replicons with associated insertion sequence (IS) elements and ESBL/PMQR genes in different farms and among several STs (e.g., IncI1-I(alpha)/IncX3) underline that plasmid transmission could be another important contributor to transmission of resistance in these farms. Conclusion: Our findings reveal a multifaceted narrative of transmission pathways. These findings could be relevant in understanding and battling the problem of antibiotic resistance in farms.

Linezolid resistance in Mycobacterium tuberculosis isolates at a tertiary care centre in Mumbai, India.

Background & objectives: Linezolid (LZD) is increasingly being used in tuberculosis (TB) treatment. However, LZD resistance has already been reported, which is highly alarming, given its critical therapeutic role. This study was aimed to phenotypically and genotypically assess LZD resistance in Mycobacterium tuberculosis (MTB) isolates at a laboratory in a tertiary care centre in Mumbai, India. Methods: A sample of 32 consecutive LZD-resistant MTB isolates identified by liquid culture susceptibility testing was subjected to whole-genome sequencing (WGS) on the Illumina NextSeq platform. Sequences were analyzed using BioNumerics software to predict resistance for 12 antibiotics within 15 min. Results: Sixty eight of the 2179 isolates tested for LZD resistance by MGIT-based susceptibility testing (June 2015 to June 2016) were LZD-resistant. Thirty two consecutive LZD-resistant isolates were analyzed by WGS to screen for known mutations conferring LZD resistance. WGS of 32 phenotypically LZD-resistant isolates showed that C154R in the rplC gene and G2814T in the rrl gene were the major resistance determinants. Interpretation & conclusions: LZD resistance poses an important risk to the success of treatment regimens, especially those designed for resistant isolates; such regimens are extensively used in India. As LZD-containing regimens increase in prominence, it is important to support clinical decision-making with an improved understanding of the common mutations conferring LZD resistance and their frequency in different settings.

Harmonization of whole-genome sequencing for outbreak surveillance of Enterobacteriaceae and Enterococci.

Whole-genome sequencing (WGS) is becoming the de facto standard for bacterial typing and outbreak surveillance of resistant bacterial pathogens. However, interoperability for WGS of bacterial outbreaks is poorly understood. We hypothesized that harmonization of WGS for outbreak surveillance is achievable through the use of identical protocols for both data generation and data analysis. A set of 30 bacterial isolates, comprising of various species belonging to the Enterobacteriaceae family and Enterococcus genera, were selected and sequenced using the same protocol on the Illumina MiSeq platform in each individual centre. All generated sequencing data were analysed by one centre using BioNumerics (6.7.3) for (i) genotyping origin of replications and antimicrobial resistance genes, (ii) core-genome multi-locus sequence typing (cgMLST) for Escherichia coli and Klebsiella pneumoniae and whole-genome multi-locus sequencing typing (wgMLST) for all species. Additionally, a split k-mer analysis was performed to determine the number of SNPs between samples. A precision of 99.0% and an accuracy of 99.2% was achieved for genotyping. Based on cgMLST, a discrepant allele was called only in 2/27 and 3/15 comparisons between two genomes, for E. coli and K. pneumoniae, respectively. Based on wgMLST, the number of discrepant alleles ranged from 0 to 7 (average 1.6). For SNPs, this ranged from 0 to 11 SNPs (average 3.4). Furthermore, we demonstrate that using different de novo assemblers to analyse the same dataset introduces up to 150 SNPs, which surpasses most thresholds for bacterial outbreaks. This shows the importance of harmonization of data-processing surveillance of bacterial outbreaks. In summary, multi-centre WGS for bacterial surveillance is achievable, but only if protocols are harmonized.

Different SARS-CoV-2 haplotypes associate with geographic origin and case fatality rates of COVID-19 patients.

The current pandemic of COVID-19 is caused by the SARS-CoV-2 virus for which many variants at the Single Nucleotide Polymorphism (SNP) level have now been identified. We show here that different allelic variants among 692 SARS-CoV-2 genome sequences display a statistically significant association with geographic origin (p < 0.000001) and COVID-19 case severity (p = 0.016). Geographic variation in itself is associated with both case severity and allelic variation especially in strains from Indian origin (p < 0.000001). Using an new alternative bioinformatics approach we were able to confirm that the presence of the D614G mutation correlates with increased case severity in a sample of 127 sequences from a shared geographic origin in the US (p = 0.018). While leaving open the question on the pathogenesis mechanism involved, this suggests that in specific geographic locales certain genotypes of the virus are more pathogenic than others. We here show that viral genome polymorphisms may have an effect on case severity when other factors are controlled for, but that this effect is swamped out by these other factors when comparing cases across different geographic regions.

Retrospective Definition of Clostridioides difficile PCR Ribotypes on the Basis of Whole Genome Polymorphisms: A Proof of Principle Study.

Clostridioides difficile is a cause of health care-associated infections. The epidemiological study of C. difficile infection (CDI) traditionally involves PCR ribotyping. However, ribotyping will be increasingly replaced by whole genome sequencing (WGS). This implies that WGS types need correlation with classical ribotypes (RTs) in order to perform retrospective clinical studies. Here, we selected genomes of hyper-virulent C. difficile strains of RT001, RT017, RT027, RT078, and RT106 to try and identify new discriminatory markers using in silico ribotyping PCR and De Bruijn graph-based Genome Wide Association Studies (DBGWAS). First, in silico ribotyping PCR was performed using reference primer sequences and 30 C. difficile genomes of the five different RTs identified above. Second, discriminatory genomic markers were sought with DBGWAS using a set of 160 independent C. difficile genomes (14 ribotypes). RT-specific genetic polymorphisms were annotated and validated for their specificity and sensitivity against a larger dataset of 2425 C. difficile genomes covering 132 different RTs. In silico PCR ribotyping was unsuccessful due to non-specific or missing theoretical RT PCR fragments. More successfully, DBGWAS discovered a total of 47 new markers (13 in RT017, 12 in RT078, 9 in RT106, 7 in RT027, and 6 in RT001) with minimum q-values of 0 to 7.40 × 10-5, indicating excellent marker selectivity. The specificity and sensitivity of individual markers ranged between 0.92 and 1.0 but increased to 1 by combining two markers, hence providing undisputed RT identification based on a single genome sequence. Markers were scattered throughout the C. difficile genome in intra- and intergenic regions. We propose here a set of new genomic polymorphisms that efficiently identify five hyper-virulent RTs utilizing WGS data only. Further studies need to show whether this initial proof-of-principle observation can be extended to all 600 existing RTs.

The phylogenetic landscape and nosocomial spread of the multidrug-resistant opportunist Stenotrophomonas maltophilia.

Recent studies portend a rising global spread and adaptation of human- or healthcare-associated pathogens. Here, we analyse an international collection of the emerging, multidrug-resistant, opportunistic pathogen Stenotrophomonas maltophilia from 22 countries to infer population structure and clonality at a global level. We show that the S. maltophilia complex is divided into 23 monophyletic lineages, most of which harbour strains of all degrees of human virulence. Lineage Sm6 comprises the highest rate of human-associated strains, linked to key virulence and resistance genes. Transmission analysis identifies potential outbreak events of genetically closely related strains isolated within days or weeks in the same hospitals.

Changes detected in the genome sequences of Escherichia coli, Listeria monocytogenes, Vibrio parahaemolyticus, and Salmonella enterica after serial subculturing.

Whole genome sequencing (WGS) is rapidly replacing other molecular techniques for identifying and subtyping bacterial isolates. The resolution or discrimination offered by WGS is significantly higher than that offered by other molecular techniques, and WGS readily allows infrequent differences that occur between 2 closely related strains to be found. In this investigation, WGS was used to identify the changes that occurred in the genomes of 13 strains of bacterial foodborne pathogens after 100 serial subcultures. Pure cultures of Shiga-toxin-producing Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Vibrio parahaemolyticus were subcultured daily for 100 successive days. The 1st and 100th subcultures were whole-genome sequenced using short-read sequencing. Single nucleotide polymorphisms (SNPs) were identified between the 1st and final culture using 2 different approaches, and multilocus sequence typing of the whole genome was also performed to detect any changes at the allelic level. The number of observed genomic changes varied by strain, species, and the SNP caller used. This study provides insight into the genomic variation that can be detected using next-generation sequencing and analysis methods after repeated subculturing of 4 important bacterial pathogens.

Epidemiological Typing of Serratia marcescens Isolates by Whole-Genome Multilocus Sequence Typing.

Serratia marcescens is an opportunistic bacterial pathogen. It is notorious for its increasing antimicrobial resistance and its potential to cause outbreaks of colonization and infections, predominantly in neonatal intensive care units (NICUs). There, its spread requires rapid infection control response. To understand its spread, detailed molecular typing is key. We present a whole-genome multilocus sequence typing (wgMLST) method for S. marcescens Using a set of 299 publicly available whole-genome sequences (WGS), we developed an initial wgMLST system consisting of 9,377 gene loci. This included 1,455 loci occurring in all reference genomes and 7,922 accessory loci. This closed system was validated using three geographically diverse collections of S. marcescens consisting of 111 clinical isolates implicated in nosocomial dissemination events in three hospitals. The validation procedure showed a full match between epidemiological data and the wgMLST analyses. We set the cutoff value for epidemiological (non)relatedness at 20 different alleles, though for the majority of outbreak-clustered isolates, this difference was limited to 4 alleles. This shows that the wgMLST system for S. marcescens provides prospects for successful future monitoring for the epidemiological containment of this opportunistic pathogen.

An Outpatient Clinic as a Potential Site of Transmission for an Outbreak of New Delhi Metallo-ß-Lactamase-producing Klebsiella pneumoniae Sequence Type 716: A Study Using Whole-genome Sequencing.

BACKGROUND: The incidence of nosocomial infections due to carbapenem-resistant Klebsiella pneumoniae is increasing worldwide. Whole-genome sequencing (WGS) can help elucidate the transmission route of nosocomial pathogens. METHODS: We combined WGS and epidemiological data to analyze an outbreak of New Delhi metallo-ß-lactamase (NDM)-producing K. pneumoniae that occurred in 2 Belgian hospitals situated about 50 miles apart. We characterized 74 NDM-producing K. pneumoniae isolates (9 from hospital A, 24 from hospital B, and 41 contemporary isolates from 15 other Belgian hospitals) using pulsed-field gel electrophoresis and WGS. RESULTS: A K. pneumoniae sequence type 716 clone was identified as being responsible for the outbreak with all 9 strains from hospital A and 20 of 24 from hospital B sharing a unique pulsotype and being clustered together at WGS (compared with 1 of 41 isolates from other Belgian hospitals). We identified the outpatient clinic of hospital B as the probable bridging site between the hospitals after combining epidemiological, phylogenetic, and resistome data. We also identified the patient who probably caused the transmission. In fact, all but 1 strain from hospital A carried a Tn1331-like transposon, whereas none of the hospital B isolates did. The patient from hospital A who did not have the Tn1331-like transposon was treated at the outpatient clinic of hospital B on the same day as the first NDM-producing K. pneumoniae-positive patient from hospital B. CONCLUSIONS: The results from our WGS-guided investigation highlight the importance of implementing adequate infection control measures in outpatient settings, especially when healthcare delivery moves from acute care facilities to outpatient clinics.

Salmonella enterica Serovar Typhi in Bangladesh: Exploration of Genomic Diversity and Antimicrobial Resistance.

Typhoid fever, caused by Salmonella enterica serovar Typhi, is a global public health concern due to increasing antimicrobial resistance (AMR). Characterization of S Typhi genomes for AMR and the evolution of different lineages, especially in countries where typhoid fever is endemic such as Bangladesh, will help public health professionals to better design and implement appropriate preventive measures. We studied whole-genome sequences (WGS) of 536 S Typhi isolates collected in Bangladesh during 1999 to 2013 and compared those sequences with data from a recent outbreak in Pakistan reported previously by E. J. Klemm, S. Shakoor, A. J. Page, F. N. Qamar, et al. (mBio 9:e00105-18, 2018, https://doi.org/10.1128/mBio.00105-18), and a laboratory surveillance in Nepal reported previously by C. D. Britto, Z. A. Dyson, S. Duchene, M. J. Carter, et al. [PLoS Negl. Trop. Dis. 12(4):e0006408, 2018, https://doi.org/10.1371/journal.pntd.0006408]. WGS had high sensitivity and specificity for prediction of ampicillin, chloramphenicol, co-trimoxazole, and ceftriaxone AMR phenotypes but needs further improvement for prediction of ciprofloxacin resistance. We detected a new local lineage of genotype 4.3.1 (named lineage Bd) which recently diverged into a sublineage (named Bdq) containing qnr genes associated with high-level ciprofloxacin resistance. We found a ceftriaxone-resistant isolate with the blaCTX-M-15 gene and a genotype distinct from the genotypes of extensively drug-resistant (XDR) isolates from Pakistan. This result suggests a different source and geographical origin of AMR. Genotype 4.3.1 was dominant in all three countries but formed country-specific clusters in the maximum likelihood phylogenetic tree. Thus, multiple independent genetic events leading to ciprofloxacin and ceftriaxone resistance took place in these neighboring regions of Pakistan, Nepal, and Bangladesh. These independent mutational events may enhance the risk of global spread of these highly resistant clones. A short-term global intervention plan is urgently needed.IMPORTANCE Typhoid fever, caused by Salmonella enterica serovar Typhi, is responsible for an estimated burden of approximately 17 million new episodes per year worldwide. Adequate and timely antimicrobial treatment invariably cures typhoid fever. The increasing antimicrobial resistance (AMR) of S Typhi severely limits the treatment options. We studied whole-genome sequences (WGS) of 536 S Typhi isolates collected in Bangladesh between 1999 and 2013 and compared those sequences with data from a recent outbreak in Pakistan and a laboratory surveillance in Nepal. The analysis suggests that multiple ancestral origins of resistance against ciprofloxacin and ceftriaxone are present in three countries. Such independent genetic events and subsequent dissemination could enhance the risk of a rapid global spread of these highly resistant clones. Given the current treatment challenges, vaccination seems to be the most appropriate short-term intervention to reduce the disease burden of typhoid fever at a time of increasing AMR.

Application of MALDI-TOF MS for the subtyping of Arcobacter butzleri strains and comparison with their MLST and PFGE types.

For the first time, this study evaluated the use of MALDI-TOF as a typing tool for Arcobacter butzleri. A total of 104 A. butzleri strains isolated from different sources in an artisanal dairy plant in Italy were identified and typed using MALDI-TOF and compared with their multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) profiles found in previous studies. MALDI-TOF correctly identified all the isolates to species level. No clearly delineated clusters appeared on dendrograms based on either the complete spectra or the significant peaks, but nine clusters were defined using the cophenetic correlation. Interestingly, MALDI-TOF proved able to discriminate A. butzleri strains below species level, confirming its potential use for epidemiological surveys. As expected, the comparative analysis with PFGE and MLST showed that the discriminatory index was lower for MALDI-TOF but roughly comparable to sequence types and pulsotypes. MALDI-TOF appears to be a relatively low cost answer to the urgent need for more rapid, less expensive typing tools suitable for source attribution studies, readily allowing multiple typing methods to be combined. This study provides insights into MALDI-TOF as potential epidemiological tool. Its application in healthcare surveillance systems awaits further exploration to encourage interaction and convergence studies between primary care in humans and animal and food veterinary authorities as part of the One Health concept.

Presence of mcr-1-positive Enterobacteriaceae in retail chicken meat but not in humans in the Netherlands since 2009.

Recently, the plasmid-mediated colistin resistance gene mcr-1 was found in Enterobacteriaceae from humans, pigs and retail meat in China. Several reports have documented global presence of the gene in Enterobacteriaceae from humans, food animals and food since. We screened several well-characterised strain collections of Enterobacteriaceae, obtained from retail chicken meat and hospitalised patients in the Netherlands between 2009 and 2015, for presence of colistin resistance and the mcr-1 gene. A total of 2,471 Enterobacteriaceae isolates, from surveys in retail chicken meat (196 isolates), prevalence surveys in hospitalised patients (1,247 isolates), clinical cultures (813 isolates) and outbreaks in healthcare settings (215 isolates), were analysed. The mcr-1 gene was identified in three (1.5%) of 196 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates from retail chicken meat samples in 2009 and 2014. Two isolates were obtained from the same batch of meat samples, most likely representing contamination from a common source. No mcr-1-positive isolates were identified among 2,275 human isolates tested. All mcr-1-positive isolates were colistin-resistant (minimum inhibitory concentration (MIC) > 2 mg/L). Our findings indicate that mcr-1-based colistin-resistance currently poses no threat to healthcare in the Netherlands. They indicate however that continued monitoring of colistin resistance and its underlying mechanisms in humans, livestock and food is needed.

Identification of beer-spoilage bacteria using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of beer-spoilage bacteria was examined. To achieve this, an extensive identification database was constructed comprising more than 4200 mass spectra, including biological and technical replicates derived from 273 acetic acid bacteria (AAB) and lactic acid bacteria (LAB), covering a total of 52 species, grown on at least three growth media. Sequence analysis of protein coding genes was used to verify aberrant MALDI-TOF MS identification results and confirmed the earlier misidentification of 34 AAB and LAB strains. In total, 348 isolates were collected from culture media inoculated with 14 spoiled beer and brewery samples. Peak-based numerical analysis of MALDI-TOF MS spectra allowed a straightforward species identification of 327 (94.0%) isolates. The remaining isolates clustered separately and were assigned through sequence analysis of protein coding genes either to species not known as beer-spoilage bacteria, and thus not present in the database, or to novel AAB species. An alternative, classifier-based approach for the identification of spoilage bacteria was evaluated by combining the identification results obtained through peak-based cluster analysis and sequence analysis of protein coding genes as a standard. In total, 263 out of 348 isolates (75.6%) were correctly identified at species level and 24 isolates (6.9%) were misidentified. In addition, the identification results of 50 isolates (14.4%) were considered unreliable, and 11 isolates (3.2%) could not be identified. The present study demonstrated that MALDI-TOF MS is well-suited for the rapid, high-throughput and accurate identification of bacteria isolated from spoiled beer and brewery samples, which makes the technique appropriate for routine microbial quality control in the brewing industry.

Reclassification of Leuconostoc gasicomitatum as Leuconostoc gelidum subsp. gasicomitatum comb. nov., description of Leuconostoc gelidum subsp. aenigmaticum subsp. nov., designation of Leuconostoc gelidum subsp. gelidum subsp. nov. and emended description of Leuconostoc gelidum.

In the present study we investigated the taxonomic status of 20 lactic acid bacteria (LAB) originating from packaged meat. On the basis of 16S rRNA gene sequence similarity, these strains were shown to belong to the genus Leuconostoc with Leuconostoc gelidum, Leuconostoc inhae and Leuconostoc gasicomitatum as the closest phylogenetic relatives. The novel strains shared more than 70 % DNA-DNA relatedness with type and reference strains of both L. gelidum and L. gasicomitatum. The DNA-DNA relatedness values between L. gelidum type and reference strains and L. gasicomitatum type and reference strains were also above 70 %, showing that all these strains belonged to the same species. Sequence analyses of concatenated atpA, pheS, and rpoA genes demonstrated that the novel strains as well as type and reference strains of L. gelidum and L. gasicomitatum are phylogenetically closely related, but form three clearly separated subgroups. Numerical analysis of HindIII ribopatterns and phenotypic tests supported this subdivision. Based on the data presented in this study, we propose to reclassify Leuconostoc gasicomitatum as Leuconostoc gelidum subsp. gasicomitatum comb. nov. (type strain, LMG 18811(T) = DSM 15947(T)). The novel strains isolated in the present study represent a novel subspecies, for which the name Leuconostoc gelidum subsp. aenigmaticum subsp. nov. is proposed, with POUF4d(T) ( = LMG 27840(T) = DSM 19375(T)) as the type strain. The proposal of these two novel subspecies automatically creates the subspecies Leuconostoc gelidum subsp. gelidum subsp. nov. (type strain, NCFB 2775(T) = DSM 5578(T)). An emended description of Leuconostoc gelidum is also provided.

Effects of growth medium on matrix-assisted laser desorption-ionization time of flight mass spectra: a case study of acetic acid bacteria.

The effect of the growth medium used on the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra generated and its consequences for species and strain level differentiation of acetic acid bacteria (AAB) were determined by using a set of 25 strains. The strains were grown on five different culture media that yielded a total of more than 600 mass spectra, including technical and biological replicates. The results demonstrate that the culture medium can have a profound effect on the mass spectra of AAB as observed in the presence and varying signal intensities of peak classes, in particular when culture media do not sustain optimal growth. The observed growth medium effects do not disturb species level differentiation but strongly affect the potential for strain level differentiation. The data prove that a well-constructed and robust MALDI-TOF mass spectrometry identification database should comprise mass spectra of multiple reference strains per species grown on different culture media to facilitate species and strain level differentiation.

Streptococcus rubneri sp. nov., isolated from the human throat.

The novel, Gram-stain-positive, ovoid, lactic acid bacterial isolates LMG 27205, LMG 27206, LMG 27207(T) and MRI-F 18 were obtained from throat samples of healthy humans. 16S rRNA gene sequence analyses indicated that these isolates belong to the genus Streptococcus, specifically the Streptococcus mitis group, with Streptococcus australis and Streptococcus mitis as the nearest neighbours (99.45 and 98.56 % 16S rRNA gene sequence similarity to the respective type strains). Genotypic fingerprinting by fluorescent amplified fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE), DNA-DNA hybridizations, comparative sequence analysis of pheS, rpoA and atpA and physiological and biochemical tests revealed that these bacteria formed a taxon well separated from its nearest neighbours and other species of the genus Streptococcus with validly published names and, therefore, represent a novel species, for which the name Streptococcus rubneri sp. nov. is proposed, with LMG 27207(T) ( = DSM 26920(T)) as the type strain.

Carnobacterium iners sp. nov., a psychrophilic, lactic acid-producing bacterium from the littoral zone of an Antarctic pond.

Two lactic acid-producing, Gram-stain-positive rods were isolated from a microbial mat actively growing in the littoral zone of an Antarctic lake (Forlidas Pond) in the Pensacola mountains and studied using a polyphasic taxonomic approach. The isolates were examined by phylogenetic analysis of the 16S rRNA gene, multilocus sequence analysis of pheS, rpoA and atpA, and biochemical and genotypic characteristics. One strain, designated LMG 26641, belonged to Carnobacterium alterfunditum and the other strain, designated LMG 26642(T), could be assigned to a novel species, with Carnobacterium funditum DSM 5970(T) as its closest phylogenetic neighbour (99.2 % 16S rRNA gene sequence similarity). Carnobacterium iners sp. nov. could be distinguished biochemically from other members of the genus Carnobacterium by the lack of acid production from carbohydrates. DNA-DNA relatedness confirmed that strain LMG 26642(T) represented a novel species, for which we propose the name Carnobacterium iners sp. nov. (type strain is LMG 26642(T)  = CCUG 62000(T)).