N2 Gas Flushing Limits the Rise of Antibiotic-Resistant Bacteria in Bovine Raw Milk during Cold Storage

Antibiotic resistance has been noted to be a major and increasing human health issue. Cold storage of raw milk promotes the thriving of psychrotrophic/psychrotolerant bacteria, which are well known for their ability to produce enzymes that are frequently heat stable. However, these bacteria also carry antibiotic resistance (AR) features. In places, where no cold chain facilities are available and despite existing recommendations numerous adulterants, including antibiotics, are added to raw milk. Previously, N-2 gas flushing showed real potential for hindering bacterial growth in raw milk at a storage temperature ranging from 6 to 25 degrees C. Here, the ability of N-2 gas (N) to tackle antibiotic-resistant bacteria was tested and compared to that of the activated lactoperoxidase system (HT) for three raw milk samples that were stored at 6 degrees C for 7 days. To that end, the mesophiles and psychrotrophs that were resistant to gentamycin (G), ceftazidime (Ce), levofloxacin (L), and trimethoprim-sulfamethoxazole (TS) were enumerated. For the log(10) ratio (which is defined as the bacterial counts from a certain condition divided by the counts on the corresponding control), classical Analyses of Variance (ANOVA) was performed, followed by a mean comparison with the Ryan-Einot-Gabriel-Welsch multiple range test (REGWQ). If the storage "time" factor was the major determinant of the recorded effects, cold storage alone or in combination with HT or with N promoted a sample-dependent response in consideration of the AR levels. The efficiency of N in limiting the increase in AR was highest for fresh raw milk and was judged to be equivalent to that of HT for one sample and superior to that of HT for the two other samples; moreover, compared to HT, N seemed to favor a more diverse community at 6 degrees C that was less heavily loaded with antibiotic multi-resistance features. Our results imply that N-2 gas flushing could strengthen cold storage of raw milk by tackling the bacterial spoilage potential while simultaneously hindering the increase of bacteria carrying antibiotic resistance/multi-resistance features.Peer reviewe

Insights into cystic fibrosis polymicrobial consortia: the role of species interactions in biofilm development, phenotype, and response to in-use antibiotics

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.02146/full#supplementary-materialCystic Fibrosis (CF) airways disease involves complex polymicrobial infections where different bacterial species can interact and influence each other and/or even interfere with the whole community. To gain insights into the role that interactions between Pseudomonas aeruginosa in co-culture with Staphylococcus aureus, Inquilinus limosus,and Stenotrophomonas maltophilia may play in infection, the reciprocal effect during biofilm formation and the response of dual biofilms toward ciprofloxacin under in vitro atmospheres with different oxygen availabilities were evaluated. Biofilm formation kinetics showed that the growth of S. aureus, I. limosus, and S. maltophilia was disturbed in the presence of P. aeruginosa, under both aerobic and anaerobic environments. On the other hand, under aerobic conditions, I. limosus led to a decrease in biofilm mass production by P. aeruginosa, although biofilm-cells viability remains unaltered. The interaction between S. maltophilia and P. aeruginosa positively influenced dual biofilm development by increasing its biomass. Compared with monocultures, biomass of P. aeruginosaC S. aureus biofilms was significantly reduced by reciprocal interference. When grown in dual biofilms with P. aeruginosa, ciprofloxacin was less effective against S. aureus, I. limosus, and S. maltophilia, with increasing antibiotic doses leading to drastic inhibitions of P. aeruginosa cultivability. Therefore, P. aeruginosa might be responsible for the protection of the whole dual consortia against ciprofloxacin activity. Based on the overall data, it can be speculated that reciprocal interferences occur between the different bacterial species in CF lung, regardless the level of oxygen. The findings also suggest that alterations of bacterial behavior due to species interplay may be important for disease progression in CF infection.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of UID/BIO/04469/2013 and COMPETE 2020 (POCI01-0145-FEDER-006684). This study was also supported by FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462), “BioHealth– Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027 and NORTE-070124-FEDER-000025 – RL2_ Environment & Health, co-funded by the Programa Operacional Regional do Norte (ON.2 – O NovoNorte), QREN, FEDER. The authors also acknowledge the grants of Susana P. Lopes (SFRH/BPD/95616/2013) and Andreia P.Magalhães (UMINHO/BD/25/2016)

Nordihydroguaiaretic acid enhances the activities of aminoglycosides against methicillin- sensitive and resistant Staphylococcus aureus in vitro and in vivo

Infections caused by methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are prevalent. MRSA infections are difficult to treat and there are no new classes of antibiotics produced to the market to treat infections caused by the resistant bacteria. Therefore, using antibiotic enhancers to rescue existing classes of antibiotics is an attractive strategy. Nordihydroguaiaretic acid (NDGA) is an antioxidant compound found in extracts from plant Larrea Tridentata. It exhibits antimicrobial activity and may target bacterial cell membrane. Combination efficacies of NDGA with many classes of antibiotics were examined by chequerboard method against 200 clinical isolates of MRSA and MSSA. NDGA in combination with gentamicin, neomycin, and tobramycin was examined by time-kill assays. The synergistic combinations of NDGA and aminoglycosides were tested in vivo using a murine skin infection model. Calculations of the fractional inhibitory concentration index (FICI) showed that NDGA when combined with gentamicin, neomycin, or tobramycin displayed synergistic activities in more than 97% of MSSA and MRSA, respectively. Time kill analysis demonstrated that NDGA significantly augmented the activities of these aminoglycosides against MRSA and MSSA in vitro and in murine skin infection model. The enhanced activity of NDGA resides on its ability to damage bacterial cell membrane leading to accumulation of the antibiotics inside bacterial cells. We demonstrated that NDGA strongly revived the therapeutic potencies of aminoglycosides in vitro and in vivo. This combinational strategy could contribute major clinical implications to treat antibiotic resistant bacterial infections

Isolation, identification and characterization of yeasts from fermented goat milk of the Yaghnob Valley in Tajikistan

The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley

Susceptibility of clinical isolates of frequently encountered bacterial species to tigecycline one year after the introduction of this new class of antibiotics: results of the second multicentre surveillance trial in Germany (G-TEST II, 2007)

Tigecycline, a broad-spectrum antibiotic for parenteral use, was introduced in Germany in May 2006. In the G-TEST-II trial, the susceptibility of isolates, recovered in 2007 from hospitalised patients in 15 centres, was assessed against tigecycline and comparators. Susceptibility tests were performed by the microdilution procedure. This study reports on the susceptibility of the isolates of 16 bacterial species and compares the results with those of a trial (G-TEST I) conducted prior to the introduction of tigecycline. Between 2005 and 2007, tigecycline retained activity against Gram-positive and Gram-negative organisms. By contrast, the rate of vancomycin-resistant strains among Enterococcus faecium isolates almost doubled. Moreover, an increase in resistance to broad-spectrum beta-lactams and fluoroquinolones was observed for members of the family Enterobacteriaceae. Against a background of a steadily rising number of pathogens that are resistant to various antibiotic classes, tigecycline represents an important treatment option

Zidovudine enhances activity of carbapenems against NDM-1-producing Enterobacteriaceae.

OBJECTIVES: To investigate the efficacy of zidovudine in combination with carbapenems against NDM-1-producing Enterobacteriaceae. METHODS: MICs were determined using the broth microdilution method. The combinatory effects of zidovudine and carbapenems were examined using the chequerboard method and time-kill analysis. RESULTS: We found that the NDM-1-producing strains were resistant to all carbapenems tested. FIC index from chequerboard assay demonstrated that zidovudine synergized with carbapenems against all the NDM-1 strains. Time-kill analysis demonstrated significant synergistic activity when a low level of zidovudine was combined with meropenem. CONCLUSIONS: Zidovudine in combination with carbapenems produced synergistic activity against NDM-1 Enterobacteriaceae strains in vitro

Low prevalence of zoonotic multidrug-resistant bacteria in veterinarians in a country with prudent use of antimicrobials in animals

The occurrence of multidrug-resistant zoonotic bacteria in animals has been increasing worldwide. Working in close contact with livestock increases the risk of carriage of these bacteria. We investigated the occurrence of extended-spectrum beta-lactamase (ESBL) and plasmidic AmpC beta-lactamase producing Enterobacteriaceae (ESBL/pAmpC-PE) and livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in Finnish veterinarians (n = 320). In addition to microbiological samples, background information was collected. Bacterial whole genome sequencing was performed to deduce sequence types (STs), spa types and resistance genes of the isolates. In total, 3.0% (9/297) of the veterinarians carried ESBL producing Escherichia coli, with one ESBL producing E. coli isolate producing also AmpC. Seven different STs, sequences of several different plasmid groups as well as several different bla(ESBL/pAmpC )genes existed in different combinations. No carbapenemase or colistin resistance genes were detected. MRSA was detected in 0.3% (1/320) of the samples. The strain belonged to LA-MRSA clonal complex (CC) 398 (ST398, spa type 011, lacking Panton-Valentine leukocidin genes). In conclusion, this study shows low carriage of multidrug-resistant zoonotic bacteria in Finnish veterinarians. However, finding LA-MRSA for the first time in a sample from a veterinarian in a country with prudent use of animal antimicrobials and regarding the recent rise of LA-MRSA on Finnish pig farms, a strong recommendation to protect people working in close contact with animals carrying LA-MRSA CC398 is given. Further studies are needed to explain why the prevalence of LA-MRSA in veterinarians is lower in Finland than in other European countries.Peer reviewe

Opening Pandora's box:High-level resistance to antibiotics of last resort in Gram-negative bacteria from Nigeria

OBJECTIVES: The aim of this study was to determine the percentage of antimicrobial-resistant isolates and the associated resistance mechanisms in Gram-negative bacteria from South Western Nigeria. METHODS: A total of 306 non-duplicate unbiased Gram-negative isolates were recovered from patients admitted to three teaching hospitals in South Western Nigeria in 2011 and 2013. Isolates were from clinical samples as well as from stool samples of inpatients without infection to assess antimicrobial resistance patterns in carriage isolates. Antimicrobial susceptibility testing was performed, and PCR and sequencing were used to identify genes encoding various known β-lactamases. Based on phenotypic and genotypic results, 10 isolates representing the diversity of phenotypes present were selected for whole-genome sequencing (WGS). RESULTS: Antimicrobial susceptibility testing revealed the following resistance rates: fluoroquinolones, 78.1%; third-generation cephalosporins, 92.2%; and carbapenems, 52.6%. More resistant isolates were isolated from stools of uninfected patients compared with clinical infection specimens. Klebsiella (10%) and Escherichia coli (7%) isolates produced a carbapenemase. WGS of selected isolates identified the presence of globally disseminated clones. CONCLUSION: This study illustrates a crisis for the use of first-line antimicrobial therapy in Nigerian patients. It is likely that Nigeria is playing a significant role in the spread of antimicrobial resistance owing to its large population with considerable global mobility