The Isolation of Vibrio crassostreae and V. cyclitrophicus in Lesser-Spotted Dogfish (Scyliorhinus canicula) Juveniles Reared in a Public Aquarium

The genus Vibrio currently contains 147 recognized species widely distributed, including pathogens for aquatic organisms. Vibrio infections in elasmobranchs are poorly reported, often with identifications as Vibrio sp. and without detailed diagnostic insights. The purpose of this paper is the description of the isolation and identification process of Vibrio spp. following a mortality event of Scyliorhinus canicula juvenile reared in an Italian public aquarium. Following investigations aimed at excluding the presence of different pathogens of marine fish species (parasites, bacteria, Betanodavirus), several colonies were isolated and subjected to species identification using the available diagnostic techniques (a biochemical test, MALDI-TOF MS, and biomolecular analysis). Discrepancies were observed among the methods; the limits of biochemistry as a unique tool for Vibrio species determination were detected through statistical analysis. The use of the rpoB gene, as a diagnostic tool, allowed the identification of the isolates as V. crassostreae and V. cyclotrophicus. Although the pathogenic role of these microorganisms in lesser-spotted dogfish juveniles has not been demonstrated, and the presence of further pathogens cannot be excluded, this study allowed the isolation of two Vibrio species in less-studied aquatic organisms, highlighting the weaknesses and strengths of the different diagnostic methods applied.

Comparative Diagnostic Accuracy of Mean and Lowest Cycle Threshold Levels in Xpert MTB/RIF Assay and Molecular Epidemiology of Missing Probes In Rifampicin Resistant Tubercular Cases From A Tertiary Care Hospital

Background: The comparative diagnostic accuracy of mean and lowest Ct values needs to be evaluated for the assessment of mycobacterial burden in tubercular cases. Mutation in any codon of 81 base-pair core regions prevents the hybridization of one or more of five overlapping Probes A-Ein Xpert MTB/RIF assay indicated by “missing probe. Molecular epidemiology of missing probes may prove useful in tracing the source of infection and selection of a more suitable drug regimen for treatment. Methods: This study included 65 rifampicin resistant cases and an equal number of rifampicin sensitive cases detected by Xpert MTB/RIF assay. Only samples tested positive for tubercular bacilli were included. The information regarding the tubercular load, Ct values of five probes targeting the rpoB gene, lowest Ct value among the five probes, missing probe in rifampicin resistant cases and time taken for the entire cycle were recorded in each case. Results: Lowest Ct is a stronger indicator of tubercular load than the mean Ct value. E probe was found to be missing in majority (64.6%) of the cases, followed by A (6.2%), B and D (4.6%), C (1.5%) probes. In 7.6% cases, more than one probe was missing. None of the probe was missing in 10.6% of rifampicin resistant cases. Conclusions: Lowest Ct value was found to be a better tool than mean Ct value for the determination of mycobacterium burden. Molecular epidemiology of missing probes could be useful in the development of new probes for the detection of rifampicin resistance.

New Insight regarding Legionella Non- Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene

Legionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non- pneumophila species identification both in clinical and environmental samples.

Molecular Characterization of Rifampicin-Resistant Staphylococcus aureus Isolates from Retail Foods in China

This study investigated the molecular characteristics of rifampin-resistant (RIF-R) Staphylococcus aureus isolates recovered from 4300 retail food samples covering most provincial capitals in China, from 2011 to 2016. Of the 1463 S. aureus enrolled, 149 isolates (142 MSSA and 7 MRSA) were identified as rifampicin-resistant, including 20 high-level (MICs ≥ 8 μg/mL) and 129 low-level (MICs between 2 and 4 μg/mL) rifampicin-resistant strains. Most of the RIF-R S. aureus isolates were resistant to more than three antibiotics. The mutations in the rifampicin resistance-determining region of the rpoB gene were studied in all RIF-R strains. All of the strains presented the mutational change 481 His/Asn and five isolates presented an additional mutation, including 477 Asp/Tyr, 527 Ile/Met, and 466 Leu/Ser, respectively. Thirteen STs and twenty-one spa types were represented, in which five MRSA showed non-type SCCmec and the remaining MRSA belonged to SCCmec type IV—where, ST1-t127 was the predominant type from all of the isolates, while ST398-t034 was the predominant type for the MRSA isolates. In this study, we found that the food-related RIF-R S. aureus may have a unique genetic background selection. However, the scenario regarding the presence of RIF-R S. aureus, especially MRSA, in retail food in China is not favorable and warrants public attention.

Аcinetobacter baumannii bv Tryptophandestruens bv nov. isolated from clinical samples

The aim of the study was to determine the taxonomic status of a group consisting of atypical strains of Acinetobacter baumannii, outline relevant characteristics and methods necessary for their identification. There were examined 10 strains of A. baumannii (6 of them primary comprised) bearing similar profile of atypical features isolated from clinical samples (urine, sputum) in 2017–2019 at the Military Medical Academy. Сlinical strains of typical A. baumannii (n = 36), Acinetobacter nosocomialis (n = 14), Acinetobacter pittii (n = 9) and 1 strain of Acinetobacter calcoaceticus isolated from the external environment were used in comparative studies. Atypical strains had the characteristics of A. calcoaceticus — A. baumannii (ACB) complex bacteria and were identified as A. baumannii. The utilization of substrates as the only carbon source was studied on a dense synthetic medium added with 0.2 % substrate during incubation for 72 hours at 37°C. Carbohydrate oxidation coupled to acid formation was detected on the Hugh–Leifson medium by using a micromethod. Aromatic amino acid biotransformation was carried out in liquid and dense nutrient media assessed in chromogenic reaction. The rpoB gene was used for strain genetic characterization. Amplification of two 940 and 1210 base pair (bp)-long fragments from the rpoB gene was performed by the routine polymerase chain reaction using primers with previously described sequences. Amplification products were sequenced by Sanger using Big Dye Terminator v3.1 (Applied Biosystems, USA) and capillary electrophoresis on an automatic sequencer ABI PRISM 3130 (Applied Biosystems, USA), followed by using methods for determining the similarity levels of sequenced fragments with the rpoB gene sequences of the reference strain A. baumannii ATCC 17978 (GenBank accession no. CP053098.1). It was found that all strains belonging to atypical A. baumannii spp. had a specific set of features that distinguish them from typical strains of A. baumannii as well as other types of the ACB complex: detected biotransformation of L-tryptophan (via anthranilate pathway) and anthranilic acid under unambiguous lack of such signs in other bacteria; lack of utilized sodium hippurate and L-arabinose being unambiguously evident in other bacteria; lack of utilized L-tryptophan, putrescine, L-ornithine being utilized in the majority of strains of belonging to other bacterial species. Genetic analysis showed that the control strains of typical A. baumannii displayed 99.20–99.21% similarity within the sequenced fragments of the rpoB gene with those from the rpoB gene of the reference strain. All 10 strains of atypical A. baumannii had similar features (99.20–99.21%). At the same time, parameters of control strains from other bacterial species significantly differed: A. nosocomialis (95.10–95.97%), A. pittii (94.63–94.92%), A. calcoaceticus (93.00%). Hence, the strains of atypical and typical A. baumannii are genetically homogeneous and belong to the same species. The data presented allow us to consider this group of atypical A. baumannii strains as a new biovar. We propose the name for this new biovar — tryptophandestruens (tryptophan-destroying) stemming from the Latin word destruens — destroying. Identification of A. baumannii bv. tryptophandestruens bacteria can be carried out in laboratory of any level by using tests for L-tryptophan biotransformation as well as sodium hippurate utilization.

Phenotypic and Molecular Characterization of Commensal, Community-Acquired and Nosocomial Klebsiella spp.

Klebsiella spp. is a relevant pathogen that can present acquired resistance to almost all available antibiotics, thus representing a serious threat for public health. While most studies have been focused on isolates causing community-acquired and nosocomial infections, little is known about the commensal isolates colonizing healthy subjects. We describe the molecular identification and the phenotypic characterization of commensal Klebsiella spp. from breast milk of healthy women and faeces from healthy breast-fed infants, which were compared with isolates from community-acquired infections and from a nosocomial NICU outbreak. The phylogenetic analysis of a 454-bp sequence of the rpoB gene was useful for species identification (K. pneumoniae, K. variicola, K. quasipneumoniae, K. oxytoca, K. grimontii, K. michiganensis, Raoultella planticola and R. ornithinolytica), previously misidentified as K. pneumoniae or K. oxytoca by biochemical methods. Globally, we report that commensal strains present virulence traits (virulence genes, siderophores and biofilms) comparable to community-acquired and NICU-infective isolates, thus suggesting that the human microbiota could constitute a reservoir for infection. Isolates causing NICU outbreak were multi-drug resistant (MDR) and ESBLs producers, although an imipenem-resistant commensal MDR K. quasipneumoniae isolate was also found. A commensal K. pneumoniae strain showed a potent bacteriocin-like inhibitory activity against MDR Klebsiella isolates, thus highlighting the potential role of commensal Klebsiella spp. in health and disease.

Molecular characterization of rpoB gene mutations in isolates from tuberculosis patients in Cubal, Republic of Angola

Background The importance of Mycobacterium tuberculosis strains with disputed rpoB mutations remains to be defined. This study aimed to assess the frequency and types of rpoB mutations in M. tuberculosis isolates from Cubal, Angola, a country with a high incidence of tuberculosis. Methods All isolates included (n = 308) were analyzed using phenotypic drug susceptibility testing and GenoType MTBDRplus assay. DNA sequencing of the rpoB gene and determination of rifampicin MIC by macrodilution method were additionally performed on isolates yielding discordant results (n = 12) and those in which the mutation detected was not characterized (n = 8). Results In total, 85.1% (74/87) of rifampicin-resistant strains had undisputed rpoB mutations -S450L (49), D435V (15), H445D (3), H445Y (2), Q432ins (1), L449M plus S450F (1), S450F (1), S450W (1) and S450Y (1)-; 10.3% (9/87) had disputed rpoB mutations—L430P plus S493L (1), N437del (1), H445L (3), D435Y (2), L452P (2)-, 2.3% (2.3%) showed no rpoB mutations and 2.3% (2/87) showed heteroresistance—D435Y plus L452P and L430P plus S493L-. Conclusion Disputed rpoB mutations were common, occurring in 10.3% of rifampicin resistant isolates. Current phenotyping techniques may be unable to detect this resistance pattern. To increase their sensitivity, a lower concentration of RIF could be used in these tests or alternatively, rpoB mutations could be screened and characterized in all M. tuberculosis strains.